Agonist anti-trk-C monoclonal antibodies

ABSTRACT

The invention concerns agonist anti-trkC monoclonal antoibodies which mimic certain biological activities of NT-3, the native ligand of trkC. The invention further concerns the use of such antibodies in the prevention and/or treatment of cellular degeneration, including nerve cell damage associated with acute nervous cell system injury and chronic neurodegenerative diseases, including peripheral neuropathy.

REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Phase of InternationalApplication PCT/US01/20153, filed Jun. 22, 2001 and claims priorityunder 35 U.S.C. §119(e) to U.S. Provisional Application Nos. 60/213,141filed Jun. 22, 2000 and 60/238,319 filed Oct. 5, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention concerns agonist anti-trkC monoclonal antibodies. Itfurther concerns the use of the agonist antibodies in the preventionand/or treatment of cellular degeneration, including nerve cell damageassociated with acute nervous cell system injury and chronicneurodegenerative diseases, including peripheral neuropathy.

2. Description of the Related Art

Neurotrophins are a family of small, basic proteins, which play acrucial role in the development and maintenance of the nervous system.The first identified and probably best understood member of this familyis nerve growth factor (NGF), which has prominent effects on developingsensory and sympathetic neurons of the peripheral nervous system(Levi-Montalcini, R. and Angeletti, P. U., Physiol. Rev. 48, 534-569[1968]; Thoenen, H. et al., Rev. Physiol. Biochem. Pharmacol. 109,145-178 [1987]). Although NGF had been known for a long time, includinga homolog from the mouse submandibular gland, the mature, active form ofwhich is often referred to as - or 2.5S NGF, it was only many yearslater that sequentially related but distinct polypeptides with similarfunctions were identified.

The first in line was a factor called brain-derived neurotrophic factor(BDNF), which was cloned and sequenced by Leibrock, J. et al. (Nature341, 149-152 [1989]). This factor was originally purified from pig brain(Barde, Y. A. et al., EMBO J. 1, 549-553 [1982]), but it was not untilits cDNA was cloned and sequenced that its homology with NGF becameapparent. The overall amino acid sequence identity between NGF and BNDFis about 50%. In view of this finding, Leibrock et al. speculated thatthere was no reason to think that BDNF and NGF should be the onlymembers of a family of neurotrophins having in common structural andfunctional characteristics.

Indeed, further neurotrophins closely related to -NGF and BDNF havesince been discovered. Several groups identified a neurotrophinoriginally called neuronal factor (NF), and now referred to asneurotrophin-3 (NT-3) (Ernfors et al., Proc. Natl. Acad. Sci. USA 87,5454-5458 (1990); Höhn et al., Nature 344, 339 [1990]; Maisonpierre etal., Science 247, 1446 [1990]; Rosenthal et al., Neuron 4, 767 [1990];Jones and Reichardt, Proc. Natl. Acad. Sci. USA 87, 8060-8064 (1990);Kaisho et al., FEBS Lett. 266, 187 [1990]. NT-3 shares about 50% of itsamino acids with both -NGF and BDNF (NT-2). Neurotrophins-4 and -5 (NT-4and NT-5), have been added to the family (U.S. Pat. No. 5,364,769 issuedNov. 15, 1994; Hallbook, F. et al., Neuron 6, 845-858 [1991]; Berkmeier,L. R. et al., Neuron 7, 857-866 [1991]; lp et al., Proc. Natl. Acad. SciUSA 89, 3060-3064 [1992]). The mammalian molecule initially described byBerkmeier et al supra, which was subsequently seen to be the homolog ofXenopus NT-4, is usually referred to as NT-4/5. In addition, there is anacidic homologous protein described in mammals which is referred to asNT-6 (Berkemeir, et al., Somat. Cell Mol. Genet 18(3):233-245 [1992]).More recently, another homologus protein from the fish, Xiphophorus hasalso been labeled NT-6 (Gotz et al., Nature 372:266-269 [1994]). Thereare two proteins described in the literature as NT-7, one cloned fromthe carp, Cyprinus, (Lai, et al., Mol. Cell. Neurosci. 11(1-2):64-76[1998]) and one from the zebrafish, Danio (Nilsson et al., FEBS Letters424(3):285-90 [1998]). None of these last three described fishneurotrophins has been described outside fish, and their relationship toany mammalian neurotrophins is unclear. The amino acid sequence ofzebrafish neurotrophin-7 (zNT-7) is more closely related to that of fishnerve growth factor (NGF) and neurotrophin-6 (NT-6) than to that of anyother neurotrophin. zNT-7 is, however, equally related to fish NGF andNT-6 (65% and 63% amino acid sequence identity, respectively) indicatingthat it represents a distinct neurotrophin sequence. zNT-7 contains a 15amino acid residue in a beta-turn region in the middle of the matureprotein. Recombinant zNT-7 was able to bind to the human p75neurotrophin receptor and to induce tyrosine phosphorylation of the rattrkA receptor tyrosine kinase, albeit less efficiently than rat NGF.zNT-7 did not interact with rat trkB or trkC, indicating a similarreceptor specificity as NGF. We propose that a diversification of theNGF subfamily in the neurotrophin evolutionary tree occurred during theevolution of teleost fishes which in the appearance of severaladditional members, such as zNT-7 and NT-6, is structurally andfunctionally related to NGF.

Neurotrophins, similarly to other polypeptide growth factors, affecttheir target cells through interactions with cell surface receptors.According to our current knowledge, two kinds of transmembraneglycoproteins serve as receptors for neurotrophins. Equilibrium bindingstudies have shown that neurotrophin-responsive neurons possess a commonlow molecular weight (65-80 kDa), low affinity receptor (LNGFR), alsotermed as p75^(NTR) or p75, which binds NGF, BDNF, and NT-3 with a K_(D)of 2×10⁻⁹ M, and large molecular weight (130-150 kDa), high affinity(K_(D) in the 10⁻¹¹ M) receptors, which are members of the trk family ofthe receptor tyrosine kinases.

The first member of the trk receptor family, trkA, was initiallyidentified as the result of an oncogenic transformation caused by thetranslocation of tropomyosin sequences onto its catalytic domain(Martin-Zanca et al., Mol. Cell. Biol. 9(1):24-33 [1989]). Later workidentified trkA as a signal transducing receptor for NGF. Subsequently,two other related receptors, mouse and rat trkB (Klein et al., EMBO J.8, 3701-3709 [1989]; Middlemas et al., Mol. Cell. Biol. 11, 143-153[1991]; EP 455,460 published 6 Nov. 1991) and porcine, mouse and rattrkC (Lamballe et al., Cell 66, 967-979 [1991]; EP 522,530 published 13Jan. 1993), were identified as members of the trk receptor family. Thestructures of the trk receptors are quite similar, but alternatesplicing increases the complexity of the family by giving rise to twoknown forms of trkA, three known forms of trkB (two without functionaltyrosine kinase domains) and at least four forms of trkC (severalwithout functional tyrosine kinase domain, and two with small inserts inthe tyrosine kinase domain).

The role of the p75 and trk receptors is controversial. It is generallyaccepted that trk receptor tyrosine kinases play an important role inconferring binding specificity to a particular neurotrophin, however,cell lines expressing trkA bind not only NGF but also NT-3 and NT-415(but not BDNF), trkB expressing cells bind BDNF, NT-3, NT-4, and NT-415(but not NGF), in contrast to trkC-expressing cells which have beenreported to bind NT-3 alone (but not the other neurotrophins).Furthermore, it has been shown in model systems that the various formsof trk receptors, arising from alternate splicing events, can activatedifferent intracellular signalling pathways, and therefore presumablymediate different physiological functions in vivo. It is unclear whethercells expressing a given trk receptor in the absence of p75 bindneurotrophins with low or high affinity (Meakin and Shooter, TrendsNeurosci. 15, 323-331 [1992]).

Published results of studies using various cell lines are confusing andsuggest that p75 is either essential or dispensable for neurotrophinresponsiveness. Cell lines that express p75 alone bind NGF, BDNF, NT-3,and NT-4 with similar low affinity at equilibrium, but the binding rateconstants are remarkably different. As a result, although p75-binding isa common property of all neurotrophins, it has been suggested the p75receptor may also play a role in ligand discrimination (Rodriguez-Tebaret al., EMBO J. 11, 917-922 [1992]). While the trk receptors have beentraditionally thought of as the biologically significant neurotrophinreceptors, it has recently been demonstrated that in melanoma cellsdevoid of trkA expression, NGF can still elicit profound changes inbiological behavior presumably through p75 (Herrmann et al., Mol. Biol.Cell 4, 1205-1216 [1993]). Davies et al. (Neuron 11, 565-574 [1993])reported the results of studies investigating the role of p75 inmediating the survival response of embryonic neurons to neurotrophins ina model of transgenic mice carrying a null mutation in the p75 gene.They found that p75 enhances the sensitivity of NGF-dependent cutaneoussensory neurons to NGF. There have now been many studies showing thatp75 is capable of mediating at least some of the biological effects ofthe neurotrophins. The field is still somewhat controversial, but p75signaling has been implicated in controlling cell death, and neuriteoutgrowth. (Barker, P A, Cell Death Diff. 5:346-356 [1998]; Bredesen etal., Cell Death Diff. 5:357-364 [1998]; Casaccia-Bonnefil, et al., CellDeath Diff. 5:357-364 [1998]; Raoul et al., Curr. Op. Neurobiol.10:111-117 [2000]; Davies, A M, Curr. Biol. 10:R198-R200 [2000]).Importantly, stimulation of p75 has been shown to modify the effects ofstimulating trkC (Hapner, et al., Developm. Biol. 201:90-100 [1998]).

The extracellular domains of full-length native trkA, trkB and trkCreceptors have five functional domains, that have been defined withreference to homologous or otherwise similar structures identified invarious other proteins. The domains have been designated starting at theN-terminus of the amino acid sequence of the mature trk receptors as 1)a first cysteine-rich domain extending from amino acid position 1 toabout amino acid position 32 of human trkA, from amino acid position 1to about amino acid position 36 of human trkB, and from amino acidposition 1 to about amino acid position 48 of human trkC; 2) aleucine-rich domain stretching from about amino acid 33 to about aminoacid to about amino acid 104 in trkA; from about amino acid 37 to aboutamino acid 108 in trkB, and from about amino acid 49 to about amino acid120 in trkC; 3) a second cysteine-rich domain from about amino acid 105to about amino acid 157 in trkA; from about amino acid 109 to aboutamino acid 164 in trkB; and from about amino acid 121 to about aminoacid 177 in trkC; 4) a first immunoglobulin-like domain stretching fromabout amino acid 176 to about amino acid 234 in trkA; from about aminoacid 183 to about amino acid 239 in trkB; and from about amino acid 196to about amino acid 257 in trkC; and 5) a second immunoglobulin-likedomain extending from about amino acid 264 to about amino acid 330 intrkA; from about amino acid 270 to about amino acid 334 in trkB; andfrom about amino acid 288 to about amino acid 351 in trkC.

Neurotrophins exhibit actions on distinct, but overlapping, sets ofperipheral and central neurons. These effects range from playing acrucial role in ensuring the survival of developing neurons (NGF insensory and sympathetic neurons) to relatively subtle effects on themorphology of neurons (NT-3 on purkinje cells). These activities haveled to interest in using neurotrophins as treatments of certainneurodegenerative diseases. NT-3 has also been found to promoteproliferation of peripheral blood leukocytes and, as a result, it hasbeen suggested that NT-3 can be used in the treatment of neutropenia,infectious disease and tumors (U.S. Pat. No. 6,015,552 issued on Jun.18, 2000).

The roles of neurotrophins in regulating cardiovascular development andmodulating the vascular response to injury have also been investigated(Donovan et al., Nature Genetics 14:210-213 [1996]; Donovan et al., A.J.Path. 147:309-324 [1995]; Kraemer et al., Arteriol. Thromb. and Vasc.Biol. 19:1041-1050 [1999]). Neurotrophins have been described aspotential therapeutics for regulating angiogenesis and vascularintegrity (PCT Publication WO 00/24415, published May 4, 2000).

Despite their promise in the treatment of cellular degeneration, such asoccurs due to neurodegenerative disease and acute neuronal injuries, andpotentially angiogenesis, neurotrophins have several shortcomings. Onesignificant shortcoming is the lack of specificity. Most neurotrophinscross-react with more than one receptor. For example NT-3, the preferredligand of the trkC receptor tyrosine kinase, also binds to and activatestrkA and trkB (Barbacid, J. Neurobiol. 25:1386-1403 [1994]; Barbarcid,Ann. New York Acad. Sci. 766:442-458 [1995]; Ryden and Ibanez, J. Biol.Chem. 271:5623-5627 [1996]; Belliveau et al., J. Cell. Biol. 136:375-388[1997]; Farinas et al., Neuron 21:325-334 [1998]). As a result, it isdifficult to devise therapies that target a specific population ofneurons. Another limitation of neurotrophin therapy is thatneurotrophins, including NT-3 are known to elicit hyperalgesia(Chaudhry, et al., Muscle and Nerve 23:189-192 [2000]). In addition,some neurotrophins such as NT-3 have poor pharmacokinetic andbioavailability properties in rodents, which raise serious questionsabout their human clinical applications (Haase et al., J. Neurol. Sci.160:S97-S105 [1998], dosages used in Helgren et al., J. Neurosci.17(1):372-82 [1997], and data below).

Accordingly, there is a great need for the development of newtherapeutic agents for the treatment of neurodegenerative disorders andacute nerve cell injuries that are devoid of the known shortcomings ofneurotrophins.

SUMMARY OF THE INVENTION

The current invention is based on the development and characterizationof agonist anti-trkC monoclonal antibodies, directed against epitopes inthe extracellular domain of trkC receptor, which mimic the biologicalactivities of NT-3, the natural ligand of trkC receptor but are free ofsome of the known detriments of NT-3. The invention also demonstratesthe usefulness of these agonist antibodies in the treatment ofneuropathy in an experimental animal model. Anti-trkC agonist antibodiesoffer numerous advantages over NT-3 in prophylactic or therapeutictreatment of cellular degeneration, such as nerve cell damage, inparticular nerve cell injury associated with neurodegenerative diseases,such as peripheral neuropathies or due to external factors, such astrauma, toxic agents, surgery, just to mention a few.

In one aspect, the invention concerns an agonist anti-trkC monoclonalantibody which

(a) shows no significant cross-reactivity with trkA or trkB; and

(b) recognizes an epitope in domain 5 of trkC.

Certain agonist antibodies of the present invention may additionallyrecognize an epitope in domain 4 of trkC. In a preferred embodiment, theantibodies bind both human and rodent (e.g. rat or mouse) trkC, and maybe murine, chimeric (including humanized) or human antibodies. Theantibodies mimic at least one activity of the native trkC ligand, NT-3,and may thus be effective in the prevention and/or treatment of variousdiseases involving cellular degeneration, including, for example,neuropathies, such as cisplatin- or pyridoxine-induced neuropathy, ordiabetic neuropathy, and (where cellular degeneration involves bonemarrow cell degeneration) disorders of insufficient blood cells, such asleukopenias including eosinopenia and/or basopenia, lymphopenia,monocytopenia, and neutropenia. In a particularly preferred embodiment,the agonist antibodies of the present invention show superior propertiesover NT-3, for example, do not cause hyperalgesia when administered to apatient, have increased bioavailability and/or higher specific activityas compared to NT-3.

In another aspect, the invention concerns an anti-trkC antibody heavychain comprising the following CDR's: a CDR1 selected from the groupconsisting of SEQ ID NOs: 1, 2, 3, 4 and 5; a CDR2 selected from thegroup consisting of SEQ ID NOs: 6, 7, 8, 9, 10 and 11; and a CDR3selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, 16 and17.

In yet another aspect, the invention concerns an anti-trkC antibodylight chain comprising the following CDR's: a CDR1 selected from thegroup consisting of SEQ ID NOs: 18, 19, 20, 21, 22, 23 and 24; a CDR2selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29 and30; and a CDR3 selected from the group consisting of SEQ ID NOs: 31, 32,33, 34, 35 and 36.

In a further aspect, the invention concerns a murine anti-trkC antibodyheavy chain comprising the following CDR's:

(a) a CDR1 of the formula XaaWXaaXaaWVK (SEQ ID NO: 37), wherein Xaa atposition 1 is F or Y; Xaa at position 3 is I or M; and Xaa at position 4is E or H;

(b) a CDR2 of the formula EIXaaPXaaXaaXaaXaaTNYNEKFKXaa (SEQ ID NO: 38),wherein Xaa at position 3 is L or Y; Xaa at position 5 is G or S; Xaa atposition 6 is S or N; Xaa at position 7 is D or G; Xaa at position 8 isN or R and Xaa at position 16 is G or S; and

(c) a CDR3 of the formula KNRNYYGNYVV (SEQ ID NO: 12) or KYYYGNSYRSWYFDV(SEQ ID NO:13).

In a still further aspect, the invention relates to a human anti-trkCantibody heavy chain comprising the following CDR's:

(a) a CDR1 of the formula XaaXaaXaaYYWXaa (SEQ ID NO: 39), wherein Xaaat position 1 is S or I; Xaa at position 2 is G or S; Xaa at position 3is G, T or Y, and Xaa at position 7 is S or N;

(b) a CDR2 of the formula XaaIXaaXaaSGSXaaTXaaNPSLKS (SEQ ID NO: 40),wherein Xaa at position 1 is Y or R; Xaa at position 3 is Y or F; Xaa atposition 4 is Y or T; Xaa at position 8 is S or R; and Xaa at position10 is N or Y; and

-   -   (c) a CDR3 of the formula selected from the group consisting of        DRDYDSTGDYYSYYGMDV (SEQ ID NO: 14); DGGYSNPFD (SEQ ID NO: 15);        ERIAAAGXaaDYYYNGLXaaV (SEQ ID NO: 41), wherein Xaa at position 8        is A or T and Xaa at position 16 is D or A.

In another aspect, the invention concerns an anti-trkC agonistmonoclonal antibody comprising a heavy chain comprising the CDR's of themurine anti-trkC antibody heavy chain of claim 14 associated with alight chain. The antibody preferably is human or comprises humanframework residues, and preferably shows no significant cross-reactivitywith trkA or trkB. Throughout the application, antibodies are defined inthe broadest sense, and specifically include antibody fragment, such asan Fv fragment, Fab fragment, Fab′ or F(ab′)₂ fragment. Antibodies ofall classes and isotypes are included, but IgG, in particular IgG-2 andIgG-4 are preferred.

In yet another aspect, the invention concerns isolated nucleic acidencoding a murine or human anti-trkC agonist antibody heavy or lightchain, or a fragment thereof. In a specific embodiment, the nucleic acidis a nucleic acid molecule deposited with ATCC on Jun. 21, 2000 under anaccession number selected from the group consisting of PTA-2133,PTA-2134, PTA-2135, PTA-2136, PTA-2137, PTA-2138, PTA-2139, PTA-2140,PTA-2141, PTA-2142 and PTA-2143.

In a further aspect, the invention concerns a vector comprising anucleic acid molecule encoding an antibody heavy and/or light chain ashereinabove defined. The invention also concerns cells transformed withsuch nucleic acid. The invention further concerns hybridoma cell linestransformed with such nucleic acid and antibodies produced by suchhybridoma cells.

In a still further aspect, the invention concerns a pharmaceuticalcomposition comprising an effective amount of an agonist anti-trkCmonoclonal antibody as hereinabove defined in admixture with apharmaceutically acceptable carrier.

In another aspect, the invention concerns a method for treating adisease or condition involving cell degeneration, comprisingadministering to a mammal an effective amount of an agonist anti-trkCantibody disclosed herein.

In yet another aspect, the invention concerns a method for treating aneuropathy or neurodegenerative disease, or repairing a damaged nervecell comprising administering to a mammal an effective amount of anagonist anti-trkC antibody disclosed herein. The neuropathy may, forexample, be a peripheral neuropathy, including, without limitation,diabetic neuropathy and large-fiber sensory neuropathies. Theneurodegenerative disease may, for example, be amyotrophic lateralsclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington'sdisease. The damaged neurons may be peripheral, such as sensory, e.g.dorsal root ganglia neurons, motor neurons, e.g. neurons from the spinalcord, or central neurons, and the injury may be due to a variety ofexternal and internal factors, including trauma, exposure toneurotoxins, metabolic diseases, infectious agents, etc.

In a further aspect, the invention concerns a method for promoting thedevelopment, proliferation, maintenance or regeneration of peripheralneurons, comprising contacting such neurons with an effective amount ofan antibody of the present invention.

In a still further aspect, the invention concerns a method for thetreatment (including prevention) of a disease or condition involvingcell degeneration in a mammalian subject by introducing nucleic acidencoding an anti-trkC antibody herein into a cell of such subject. Themethod (gene therapy) preferably concerns the treatment of a neuropathyor neurodegenerative disease, or reparation of a damaged nerve cell.Accordingly, the recipient cells preferably are nerve cells.

In yet another aspect, the invention concerns delivery vehiclescontaining genetic material (nucleic acid) encoding an anti-trkCantibody suitable for gene therapy use.

In an additional aspect, the invention concerns a method of inducingangiogenesis by delivering an anti-trkC antibody of the presentinvention in an amount effective to induce angiogenesis. The deliveryspecifically includes the administration of the antibodies and thedelivery of nucleic acid encoding the antibodies (e.g. in gene therapy).

In yet another aspect, the invention concerns an isolated nucleic acidmolecule encoding a murine or human anti-trkC agonist antibody heavy orlight chain selected from the group consisting of SEQ ID NO: 58, SEQ IDNO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQID NO: 64, SEQ ID NO: 65; SEQ ID NO: 66; SEQ ID NO: 67, SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71. The present inventionalso concerns a polypeptide encoded by one or more of the isolatednucleic acid molecules.

In another aspect, the invention concerns a whole cell transformed withnucleic acid encoding murine or human anti-trkC agonist antibody heavychain, light chain or both heavy and light chain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D show agonist activity of various human (A and C) and murine(B and D) monoclonal antibodies against trkC receptor demonstrated usingKIRA (A and B) and PC12 neurite outgrowth assay (C and D). Protein Apurified monoclonal antibodies were diluted to 27 μg/ml in KIRAstimulation buffer (F12/DMEM 50:50 containing 2% bovine serum albumin[BSA, Intergen Co., Purchase, N.Y.) and 25 mM Hepes, 0.2 μm filtered).The monoclonal antibodies were then diluted 1:3 (8 dilutions total;concentrations ranged from 0.01-180 nM Nab) in stimulation media.GD-transfected CHO cells (5×10⁴ cells/well) were then stimulated witheither NT-3 or Mab (dilutions assayed in duplicate) for 6 hours and theassay was completes as described in the examples (FIG. 1A, human Mabs;FIG. 1B, murine Mabs). The purified Mabs were assayed for agonistactivity in the PC12 neurite outgrowth assay as described in theexamples. Rat PC12 cells were transfected with full-length human trkCand the cells plated at a density of 1000 cells/well. Three daysfollowing transfection, the Mabs were added in triplicate(concentrations ranging from 0.0002 to 2.7 nM) to the wells containingthe trkC transfectants and incubated for an additional 3 days at 37° C.The cells were then analyzed by phase contrast microscopy and cells withneurites exceeding two-times the diameter of the cell were counted.

FIG. 2 shows that agonist anti-trkC monoclonal antibodies bindspecifically to trkC using 6.1.2 antibody as a representative example.

FIG. 3 demonstrates that agonist anti-trkC monoclonal antibodiesrecognize human trkC more efficiently than rat trkC. The ability of themonoclonal antibodies to bind rat trkC was determined using animmunoadhesin construct of the receptor. TrkC (human trkC-gD or rattrkC-IgG) was immobilized on microtiter plates (100 μl of a 1 μg/mlsolution diluted in 50 mM carbonate buffer, pH 9.5) overnight. Theplates were washed and blocked. The Mabs were then diluted to 1 μg/ml inPBS containing 0.5% BSA and 0.05% Tween 20, added to the appropriatewells (100 μl/well), and incubated for one hour at room temperature. Theplates were washed and the appropriate HRP conjugate was added (humanMabs: goat anti-human κ-HRP, 1:5K; murine Mabs: goat anti-molgG(Fc)-HRP, 1:5 K) and incubated for one hour at room temperature. Theplates were then washed, developed and read.

FIG. 4 shows a representative example of epitope mapping usingcompetition ELISA. A biotinylated human anti-trkC 6.1.2 monoclonalantibody was incubated with immobilized trkC in the absence or presenceof excess of various unlabeled anti-trkC monoclonal antibodies.

FIG. 5 summarizes the results of epitope mapping using competitionELISA.

FIGS. 6A-C show a schematic diagram of various trkC chimera (A) andtheir use in mapping of epitopes on trkC recognized by various agonisthuman (B) and murine (C) anti-trkC monoclonal antibodies.

FIG. 7 shows amino acid sequence of human trkC domain 4 and 5 showingresidues that were targeted for mutagenesis to decipher their roles inrecognition by agonist anti-trkC monoclonal antibodies.

FIG. 8 shows 3-dimensional ribbon diagram of trkC in complex withanti-trkC monoclonal antibodies. Specifically shown are the amino acidresidues of trkC that are likely to play an important role inrecognition by CDRs of anti-trkC antibodies.

FIG. 9 shows the amino acid sequence of the heavy chain variable (V_(H))region from murine and human anti-trkC agonist monoclonal antibodies. Inaddition, the three CDR regions (CDR1, CDR2 and CDR3) are highlighted inbold. The amino acid sequence of CDR1 of the 2250 and 2253 heavy chainis SEQ ID NO: 1. The amino acid sequence of CDR1 of the 2256 heavy chainis SEQ ID NO: 2. The amino acid sequence of CDR1 of the 6.1.2 and 2345heavy chain is SEQ ID NO: 3. The amino acid sequence of CDR1 of the6.4.1 heavy chain is SEQ ID NO: 4. The amino acid sequence of CDR1 ofthe 2349 heavy chain is SEQ ID NO: 5. The amino acid sequence of CDR2 ofthe 2250 and 2253 heavy chain is SEQ ID NO: 6. The amino acid sequenceof CDR2 of the 2256 heavy chain is SEQ ID NO: 7. The amino acid sequenceof CDR2 of the 6.1.2 heavy chain is SEQ ID NO: 8. The amino acidsequence of CDR2 of the 6.4.1 heavy chain is SEQ ID NO: 9. The aminoacid sequence of CDR2 of the 2345 heavy chain is SEQ ID NO: 10. Theamino acid sequence of CDR2 of the 2349 heavy chain is SEQ ID NO: 11.The amino acid sequence of CDR3 of the 2250 and 2253 heavy chain is SEQID NO: 12. The amino acid sequence of CDR3 of the 2256 heavy chain isSEQ ID NO: 13. The amino acid sequence of CDR3 of the 6.1.2 heavy chainis SEQ ID NO: 14. The amino acid sequence of CDR3 of the 6.4.1 heavychain is SEQ ID NO: 15. The amino acid sequence of CDR3 of the 2345heavy chain is SEQ ID NO: 16. The amino acid sequence of CDR3 of the2349 heavy chain is SEQ ID NO: 17.

FIG. 10 shows the amino acid sequence of the light chain variable(V_(L)) region from murine and human anti-trkC agonist monoclonalantibodies. In addition, the three CDR regions (CDR1, CDR2 and CDR3) arehighlighted in bold. The amino acid sequence of CDR1 of the 2250 lightchain is SEQ ID NO: 18. The amino acid sequence of CDR1 of the 2253light chain is SEQ ID NO: 19. The amino acid sequence of CDR1 of the2256 light chain is SEQ ID NO: 20. The amino acid sequence of CDR1 ofthe 6.1.2 light chain is SEQ ID NO: 21. The amino acid sequence of CDR1of the 6.4.1 light chain is SEQ ID NO: 22. The amino acid sequence ofCDR1 of the 2345 light chain is SEQ ID NO: 23. The amino acid sequenceof CDR1 of the 2349 light chain is SEQ ID NO: 24. The amino acidsequence of CDR2 of the 2250 light chain is SEQ ID NO: 25. The aminoacid sequence of CDR2 of the 2253 light chain is SEQ ID NO: 26. Theamino acid sequence of CDR2 of the 2256 light chain is SEQ ID NO: 27.The amino acid sequence of CDR2 of the 6.1.2 light chain is SEQ ID NO:28. The amino acid sequence of CDR2 of the 6.4.1 light chain is SEQ IDNO: 29. The amino acid sequence of CDR2 of the 2345 and 2349 light chainis SEQ ID NO: 30. The amino acid sequence of CDR3 of the 2250 lightchain is SEQ ID NO: 31. The amino acid sequence of CDR3 of the 2253light chain is SEQ ID NO: 32. The amino acid sequence of CDR3 of the2256 light chain is SEQ ID NO: 33. The amino acid sequence of CDR3 ofthe 6.1.2 light chain is SEQ ID NO: 34. The amino acid sequence of CDR3of the 6.4.1 light chain is SEQ ID NO: 35. The amino acid sequence ofCDR3 of the 2345 and 2349 light chain is SEQ ID NO: 36.

FIG. 11 shows amino acid sequence of CDRs of heavy and light variablechains of murine and human anti-trkC agonist monoclonal antibodies. Alsoshown are the families to which these sequences belong based on homologywith CDR sequences available in databases.

FIG. 12 shows that anti-trkC agonist monoclonal antibodies have improvedhalf-life and bioavailability in vivo.

FIG. 13 shows effect of anti-trkC agonist monoclonal antibodies oncisplatin-induced neuropathy.

FIG. 14 shows decrease in marker expression caused by pyridoxineneuropathy.

FIG. 15 shows amelioration of the effects of low doses of pyridoxine byagonist anti-trkC monoclonal antibodies.

FIG. 16 shows amelioration of the effects of high doses of pyridoxine byagonist anti-trkC monoclonal antibodies.

FIG. 17 shows amelioration of pyridoxine neuropathy by an anti-trkCagonist monoclonal antibody.

FIG. 18 shows attenuation of pyridoxine-induced deficit of ladder byagonist anti-trkC monoclonal antibodies.

FIG. 19 shows that NT3, but not anti-trkC agonist monoclonal antibodies,causes hyperalgesi at therapeutic doses.

FIG. 20 shows the amino acid sequence of human trkC receptor (SEQ ID NO:56) where the boundaries of domains 4 and 5 are indicated.

FIG. 21 (in 2 pages) shows the nucleotide sequence of human trkCreceptor (SEQ ID NO: 57).

FIG. 22 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:58) and light chain (B; SEQ ID NO: 59) of the anti-trkC agonistmonoclonal antibody 2250.

FIG. 23 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:60) and light chain (B; SEQ ID NO: 61) of the anti-trkC agonistmonoclonal antibody 2253.

FIG. 24 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:62) and light chain (B; SEQ ID NO: 63) of the anti-trkC agonistmonoclonal antibody 2256.

FIG. 25 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:64) and light chain (B; SEQ ID NO: 65) of the anti-trkC agonistmonoclonal antibody 2345.

FIG. 26 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:66) and light chain (B; SEQ ID NO: 67) of the anti-trkC agonistmonoclonal antibody 2349.

FIG. 27 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:68) and light chain (B; SEQ ID NO: 69) of the anti-trkC agonistmonoclonal antibody 6.1.2.

FIG. 28 shows the nucleotide sequence of the heavy chain (A; SEQ ID NO:70) and light chain (B; SEQ ID NO: 71) of the anti-trkC agonistmonoclonal antibody 6.4.1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Definitions

The term “neurotrophin” and its grammatical variants are usedinterchangeably, and refer to a family of polypeptides comprising nervegrowth factor (NGF) and sequentially related homologs. NGF,brain-derived growth factor (BDNF, a.k.a. NT-2), neurotrophin-3 (NT-3),neurotrophins-4 and -5 (NT-415), neurotrophin-6 (NT-6), andneurotrophin-7 (NT-7) have so far been identified as members of thisfamily.

The term “neurotrophin” includes native neurotrophins of any (human ornon-human) animal species, and their functional derivatives, whetherpurified from a native source, prepared by methods of recombinant DNAtechnology, or chemical synthesis, or any combination of these or othermethods. “Native” or “native sequence” neurotrophins have the amino acidsequence of a neurotrophin occurring in nature in any human or non-humananimal species, including naturally-occurring truncated and variantforms, and naturally-occurring allelic variants.

The terms “trk”, “trk polypeptide”, “trk receptor” and their grammaticalvariants are used interchangeably and refer to polypeptides of thereceptor tyrosine kinase superfamily, which are capable of binding atleast one native neurotrophin. Currently identified members of thisfamily are trkA (p140^(trkA)), trkB, and trkC.

The expression “extracellular domain” or “ECD” when used herein refersto any polypeptide sequence that shares a ligand binding function of theextracellular domain of a naturally occurring receptor. Ligand bindingfunction of the extracellular domain refers to the ability of thepolypeptide to bind to a ligand. Accordingly, it is not necessary toinclude the entire extracellular domain since smaller segments have beenfound to be adequate for ligand binding. The truncated extracellulardomain is generally soluble. The term ECD encompasses polypeptidesequences in which the hydrophobic transmembrane sequence (and,optionally, 1-20 amino acids C-terminal and/or N-terminal to thetransmembrane domain) of the mature receptor has been deleted.

The term “agonist anti-trkC antibody” refers to an antibody, which isable to bind to and activate a native sequence trkC receptor and/ordownstream pathways mediated by the trkC signaling function therebymimicking a biological activity of a native ligand of the receptor, inparticular NT-3. For example, the agonist antibody may bind to the ECDdomain of a trkC receptor and thereby cause dimerization of thereceptor, resulting in activation of the intracellular catalytic kinasedomain. Consequently, this may result in stimulation of growth and/ordifferentiation of cells expressing the receptor in vitro and/or invivo. The agonist antibodies of the present invention preferablyrecognize an epitope that includes at least part of domain 5 (amino acidpositions from about 266 to about 381) and/or domain 4 (amino acidposition from about 178 to about 265) of the human trkC receptor or acorresponding epitope on a non-human, e.g. murine trkC receptor.

“Biological activity”, when used in conjunction with the agonistanti-trkC antibodies of the present invention, generally refers tohaving an effector function in common with NT-3, the native ligand oftrkC. The effector function preferably is the ability to bind andactivate the trkC receptor tyrosine kinase and/or downstream pathwaysmediated by the trkC signaling function. Without limitation, preferredbiological activities include the ability to promote the development,proliferation, maintenance and/or regeneration of damaged cells, inparticular neurons in vitro or in vivo, including peripheral(sympathetic, parasympathetic, sensory, and enteric) neurons,motorneurons, and central (brain and spinal cord) neurons, andnon-neuronal cells, e.g. peripheral blood leukocytes. A particularlypreferred biological activity is the ability to treat (includingprevention) a neuropathy, e.g. peripheral neuropathy or otherneurodegenerative disease, or repair a damaged nerve cell. The damagedneurons may be sensory, sympathetic, parasympathetic, or enteric, e.g.dorsal root ganglia neurons, motorneurons, and central neurons, e.g.neurons from the spinal cord, and the damage may be of any cause,including trauma, toxic agents, surgery, stroke, ischemia, infection,metabolic disease, nutritional deficiency, and various malignancies.Another specific biological activity is the ability to induceangiogenesis.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. Specifically, the treatment may directlyprevent, slow down or otherwise decrease the pathology of cellulardegeneration of damage, such as the pathology of nerve cells, or mayrender the cells, e.g. neurons more susceptible to treatment by othertherapeutic agents. In a preferred embodiment, the treatment reduces orslows down the decline and/or stimulates the restoration of the functionof target neurons.

The “pathology” of a (chronic) neurodegenerative disease or acutenervous system injury includes all phenomena that affect the well beingof the patient including, without limitation, neuronal disfunction,degeneration, injury and/or death.

The terms “neurodegenerative disease” and “neurodegenerative disorder”are used in the broadest sense to include all disorders the pathology ofwhich involves neuronal degeneration and/or disfunction, including,without limitation, peripheral neuropathies; motorneuron disorders, suchas amylotrophic lateral schlerosis (ALS, Lou Gehrig's disease), Bell'spalsy, and various conditions involving spinal muscular atrophy orparalysis; and other human neurodegenerative diseases, such asAlzheimer's disease, Parkinson's disease, epilepsy, multiple schlerosis,Huntington's chorea, Down's Syndrome, nerve deafness, and Meniere'sdisease.

“Peripheral neuropathy” is a neurodegenerative disorder that affects theperipheral nerves, most often manifested as one or a combination ofmotor, sensory, sensorimotor, or autonomic dysfunction. Peripheralneuropathies may, for example, be genetically acquired, can result froma systemic disease, or can be induced by a toxic agent, such as aneurotoxic drug, e.g. antineoplastic agent, or industrial orenvironmental pollutant. “Peripheral sensory neuropathy” ischaracterized by the degeneration of peripheral sensory neurons, whichmay be idiopathic, may occur, for example, as a consequence of diabetes(diabetic neuropathy), cytostatic drug therapy in cancer (e.g. treatmentwith chemotherapeutic agents such as vincristine, cisplatin,methotrexate, 3′-azido-3′-deoxythymidine, or taxanes, e.g. paclitaxel[TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.] and doxetaxel[TAXOTERE®, Rhône-Poulenc Rorer, Antony, France]), alcoholism, acquiredimmunodeficiency syndrom (AIDS), or genetic predisposition. Geneticallyacquired peripheral neuropathies include, for example, Refsum's disease,Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease,Dejerine-Sottas syndrome, Abetalipoproteinemia, and Charcot-Marie-Tooth(CMT) Disease (also known as Proneal Muscular Atrophy or HereditaryMotor Sensory Neuropathy (HMSN)). Most types of peripheral neuropathydevelop slowly, over the course of several months or years. In clinicalpractice such neuropathies are called chronic. Sometimes a peripheralneuropathy develops rapidly, over the course of a few days, and isreferred to as acute. Peripheral neuropathy usually affects sensory andmotor nerves together so as to cause a mixed sensory and motorneuropathy, but pure sensory and pure motor neuropathy are also known.

The term “toxic agent”, as used in the context of the present invention,is meant to refer to a substance that, through its chemical action,injures, impairs, or inhibits the activity of a component of the nervoussystem. The long list of toxic agents (also referred to as “neurotoxicagents”) includes, without limitation, chemotherapeutic agents, such asthose listed above, alcohol, metals, industrial toxins, contaminants offood and medicines, etc.

“Mammal” for purpose of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sport orpet animals, such as dogs, horses, sheep, cats, cows, etc. Preferably,the mammal is human.

The term “trkC immunoadhesin” is used interchangeably with theexpression “trkC-immunoglobulin chimera” and refers to a chimericmolecule that combines a portion of trkC (generally the extracellulardomain thereof) with an immunoglobulin sequence. The immunoglobulinsequence preferably, but not necessarily, is an immunoglobulin constantdomain. Chimeras constructed from a receptor sequence linked to anappropriate immunoglobulin constant domain sequence (immunoadhesins) areknown in the art. Immunoadhesins reported in the literature includefusions of the T cell receptor* (Gascoigne et al., Proc. Natl. Acad.Sci. USA, 84: 2936-2940 [1987]); CD4* (Capon et al., Nature 337: 525-531[1989]; Traunecker et al., Nature, 339: 68-70 [1989]; Zettmeissl et al.,DNA Cell Biol. 9: 347-353 [1990]; Byrn et al., Nature, 344: 667-670[1990]); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.,110:2221-2229 [1990]; Watson et al., Nature, 349: 164-167 [1991]); CD44*(Aruffo et al., Cell, 61: 1303-1313 [1990]; CD28* and B7* (Linsley etal., J. Exp. Med., 173: 721-730 [1991]); CTLA-4* (Lisley et al., J. Exp.Med. 174: 561-569 [1991]; CD22* (Stamenkovic et al., Cell, 66:1133-11144[1991]); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88:10535-10539 [1991]; Lesslauer et al., Eur. J. Immunol, 27:2883-2886[1991]; Peppel et al., J. Exp. Med., 174:1483-1489 [1991]); NP receptors(Bennett et al., J. Biol. Chem. 266:23060-23067 [1991]); and IgEreceptor α* (Ridgway et al., J. Cell. Biol., 115:abstr. 1448 [1991]),where the asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily.

“Isolated” nucleic acid or polypeptide in the context of the presentinvention is a nucleic acid or polypeptide that is identified andseparated from contaminant nucleic acids or polypeptides present in theanimal or human source of the nucleic acid or polypeptide. The nucleicacid or polypeptide may be labeled for diagnostic or probe purposes,using a label as described and defined further below in discussion ofdiagnostic assays.

In general, the term “amino acid sequence variant” refers to moleculeswith some differences in their amino acid sequences as compared to areference (e.g. native sequence) polypeptide. The amino acid alterationsmay be substitutions, insertions, deletions or any desired combinationsof such changes in a native amino acid sequence.

The terms “DNA sequence encoding”, “DNA encoding” and “nucleic acidencoding” refer to the order or sequence of deoxyribonucleotides along astrand of deoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide chain. The DNAsequence thus codes for the amino acid sequence.

The terms “replicable expression vector” and “expression vector” referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancer.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

In the context of the present invention the expressions “cell”, “cellline”, and “cell culture” are used interchangeably, and all suchdesignations include progeny. Thus, the words “transformants” and“transformed (host) cells” include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological activity as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

An “exogenous” element is defined herein to mean nucleic acid sequencethat is foreign to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is ordinarily notfound.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework region (FR). The variabledomains of native heavy and light chains each comprise four FRs (FR1,FR2, FR3 and FR4, respectively), largely adopting a -sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the -sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647-669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (i.e. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain: Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa ( ) and lambda ( ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called , , , , and , respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antibody” herein is used in the broadest sense andspecifically covers human, non-human (e.g. murine) and humanizedmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Reichmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The expression “linear antibodies” when used throughout this applicationrefers to the antibodies described in Zapata et al. Protein Eng.8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens.

Antibodies which bind to domain 5 and/or 4 within the amino acidsequence of native sequence human trkC, or to an equivalent epitope in anative sequence non-human trkC receptor, are identified by “epitopemapping.” There are many methods known in the art for mapping andcharacterizing the location of epitopes on proteins, including solvingthe crystal structure of an antibody-antigen complex, competitionassays, gene fragment expression assays, and synthetic peptide-basedassays, as described, for example, in Chapter 11 of Harlow and Lane,Using Antibodies, a Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1999. A competition ELISA assay isspecifically described in Example 1. According to the gene fragmentexpression assays, the open reading frame encoding the protein isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of the protein with the antibodyto be tested is determined. The gene fragments may, for example, beproduced by PCR and then transcribed and translated into protein invitro, in the presence of radioactive amino acids. The binding of theantibody to the radioactively labeled protein fragments is thendetermined by immunoprecipitation and gel electrophoresis. Certainepitopes can also be identified by using large libraries of randompeptide sequences displayed on the surface of phage particles (phagelibraries). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays. The latter approach is suitable to define linearepitopes of about 5 to 15 amino acids.

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels. A competitionELISA assay is disclosed in Example 1.

The term amino acid or amino acid residue, as used herein, refers tonaturally occurring L amino acids or to D amino acids as describedfurther below with respect to variants. The commonly used one- andthree-letter abbreviations for amino acids are used herein (BruceAlberts et al., Molecular Biology of the Cell, Garland Publishing, Inc.,New York (3d ed. 1994)).

Hybridization is preferably performed under “stringent conditions” whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50 C, or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42 C. Another example is use of50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 618), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42 C, with washes at 42 C in 0.2×SSC and 0.1% SDS.

B. Methods for Carrying Out the Invention

The present invention concerns agonist human and non-human monoclonalantibodies (including humanized forms of the latter), which mimickcertain biological properties of NT-3, the native ligand of the trkCreceptor. General techniques for the production of murine and humananti-trkC antibodies are well known in the art and are describedhereinbelow. Further details, including the selection of agonistantibodies, are provided in Example 1.

1. Antibody Preparation

(i) Polyclonal Antibodies

Methods of preparing polyclonal antibodies are known in the art.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. It may beuseful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized, such as serum albumin, orsoybean trypsin inhibitor. Examples of adjuvants which may be employedinclude Freund's complete adjuvant and MPL-TDM.

(ii) Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103, [Academic Press, 1986]).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), conditions under which the growth ofHGPRT-deficient cells is prevented.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and M.C.-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp.51-63,Marcel Dekker, Inc., New York, [1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, DMEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison, et al., Proc. Nat.Acad. Sci. 81, 6851 (1984), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of an anti-trkmonoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for an trkreceptor and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Recombinant production of antibodies will be described in more detailbelow.

(iii) Humanized Antibodies

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a non-human source. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers [Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody.

Accordingly, such “humanized” antibodies are chimeric antibodies(Cabilly, supra), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed 21 August 192,which is a continuation-in-part of application Ser. No. 07/715,272 filed14 Jun. 1991.

(iv) Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp.51-63 (Marcel Dekker, Inc.,New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al.,Nature 362, 255-258 (1993).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, the phage display technology (McCafferty et al., Nature348, 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimicks someof the properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993),and the isolation of a high affinity human antibody directly from suchlarge phage library is reported by Griffith et al., EMBO J. (1994), inpress. Gene shuffling can also be used to derive human antibodies fromrodent antibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published 1 Apr. 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

(v) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe trkC receptor to provide an agonist antibody, the other one is forany other antigen, and preferably for another receptor or receptorsubunit. For example, bispecific antibodies specifically binding a trkCreceptor and a neurotrophin, or a trkC receptor and another trk receptorare within the scope of the present invention.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published 13 May 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2 and CH3 regions. Itis preferred to have the first heavy chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are cotransfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inPCT Publication No. WO 94/04690, published on Mar. 3, 1994.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(vi) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

(vii) Antibody Fragments

In certain embodiments, the anti-trkC antibody (including murine, humanand humanized antibodies, and antibody variants) is an antibodyfragment. Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al.,Science 229:81 (1985)). However, these fragments can now be produceddirectly by recombinant host cells. For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Inanother embodiment, the F(ab′)₂ is formed using the leucine zipper GCN4to promote assembly of the F(ab′)₂ molecule. According to anotherapproach, Fv, Fab or F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner.

(viii) Amino Acid Sequence Variants of Antibodies

Amino acid sequence variants of the anti-trkC antibodies are prepared byintroducing appropriate nucleotide changes into the anti-trkC antibodyDNA, or by peptide synthesis. Such variants include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the anti-trkC antibodies of theexamples herein. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of the humanized orvariant anti-trkC antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-trkC antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells Science, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with trkC antigen. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-trkCantibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-trkC antibody with an N-terminal methionyl residue or theantibody fused to an epitope tag. Other insertional variants of theanti-trkC antibody molecule include the fusion to the N- or C-terminusof the anti-trkC antibody of an enzyme or a polypeptide which increasesthe serum half-life of the antibody (see below).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-trkC antibodymolecule removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Exemplary Preferred Original Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn;glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; argarg Ile (I) leu; val; met; ala; leu phe; norleucine Leu (L) norleucine;ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe;ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thrthr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; met; phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the humanized or variant anti-trkC antibody also may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant crosslinking. Conversely, cysteine bond(s)may be added to the antibody to improve its stability (particularlywhere the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants is affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. binding affinity) as herein disclosed. Inorder to identify candidate hypervariable region sites for modification,alanine scanning mutagenesis can be performed to identify hypervariableregion residues contributing significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and human trkC. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

(ix) Glycosylation Variants of Antibodies

Antibodies are glycosylated at conserved positions in their constantregions (Jefferis and Lund, Chem. Immunol. 65:111-128 [1997]; Wright andMorrison, TibTECH 15:26-32 [1997]). The oligosaccharide side chains ofthe immunoglobulins affect the protein's function (Boyd et al., Mol.Immunol. 32:1311-1318 [1996]; Wittwe and Howard, Biochem. 29:4175-4180[1990]), and the intramolecular interaction between portions of theglycoprotein which can affect the conformation and presentedthree-dimensional surface of the glycoprotein (Hefferis and Lund, supra;Wyss and Wagner, Current Opin. Biotech. 7:409-416 [1996]).Oligosaccharides may also serve to target a given glycoprotein tocertain molecules based upon specific recognition structures. Forexample, it has been reported that in agalactosylated IgG, theoligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminalN-acetylglucosamine residues become available to bind mannose bindingprotein (Malhotra et al., Nature Med. 1:237-243 [1995]). Removal byglycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinanthumanized murine monoclonal IgG1 antibody which recognizes the CDw52antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO)cells resulted in a complete reduction in complement mediated lysis(CMCL) (Boyd et al., Mol. Immunol. 32:1311-1318 [1996]), while selectiveremoval of sialic acid residues using neuraminidase resulted in no lossof DMCL. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., Mature Biotech.17:176-180 [1999]).

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc. Glycosylation variants may, for example, be preparedby removing, changing and/or adding one or more glycosylation sites inthe nucleic acid sequence encoding the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of theanti-trkC antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-trkC antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the underlying nucleotide sequence.Glycosylation largely depends on the host cell used to express theantibody. Since the cell type used for expression of recombinantglycoproteins, e.g. antibodies, as potential therapeutics is rarely thenative cell, significant variations in the glycosylation pattern of theantibodies can be expected (see, e.g. Hse et al., J. Biol. Chem.272:9062-9070 [1997]). In addition to the choice of host cells, factorswhich affect glycosylation during recombinant production of antibodiesinclude growth mode, media formulation, culture density, oxygenation,pH, purification schemes and the like. Various methods have beenproposed to alter the glycosylation pattern achieved in a particularhost organism including introducing or overexpressing certain enzymesinvolved in oligosaccharide production (U.S. Pat. Nos. 5,047,335;5,510,261 and 5,278,299). Glycosylation, or certain types ofglycosylation, can be enzymatically removed from the glycoprotein, forexample using endoglycosidase H (Endo H). In addition, the recombinanthost cell can be genetically engineered, e.g. make defective inprocessing certain types of polysaccharides. These and similartechniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

(x) Other Modifications of Antibodies

The anti-trkC antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Aced Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci.USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.81(19):1484 (1989).

The antibody of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as -galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; -lactamaseuseful for converting drugs derivatized with -lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-trkCantibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604-608 [1984]).

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody. In this case, it maybe desirable to modify the antibody fragment in order to increase itsserum half-life. This may be achieved, for example, by incorporation ofa salvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis). See WO96/32478 published Oct. 17, 1996.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the CL region or V_(L)region, or both, of the antibody fragment.

Covalent modifications of the humanized or variant anti-trkC antibody(including glycosylation variants) are also included within the scope ofthis invention. They may be made by chemical synthesis or by enzymaticor chemical cleavage of the antibody, if applicable. Other types ofcovalent modifications of the antibody are introduced into the moleculeby reacting targeted amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues. Exemplary covalent modifications ofpolypeptides are described in U.S. Pat. No. 5,534,615, specificallyincorporated herein by reference. A preferred type of covalentmodification of the antibody comprises linking the antibody to one of avariety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

2. Vectors, Host Cells and Recombinant Methods

The invention also provides isolated nucleic acid encoding thenon-human, e.g. murine and human anti-trkC antibodies of the presentinvention (including the humanized versions of the non-humanantibodies), vectors and host cells comprising the nucleic acid, andrecombinant techniques for the production of the antibodies.

For recombinant production of an antibody, the nucleic acid encoding itmay be isolated and inserted into a replicable vector for furthercloning (amplification of the DNA) or for expression. In anotherembodiment, the antibody may be produced by homologous recombination,e.g. as described in U.S. Pat. No. 5,204,244, specifically incorporatedherein by reference. DNA encoding the monoclonal antibody is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody). Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, e.g., as described in U.S.Pat. No. 5,534,615 issued Jul. 9, 1996 and specifically incorporatedherein by reference.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Sarratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E coliX1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. Theseexamples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-trkCantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045) K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated anti-trkCantibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol 36:59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216[1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251[1980]); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 [1982]); MRC 5 cells; and FS4 cells.

Host cells are transformed with the above-described expression orcloning vectors for anti-trkC antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

The host cells used to produce the anti-trkC antibody of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM) (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human 1, 2, or 4 heavychains (Lindmark et al., J. Immunol Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human 3 (Guss et al., EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attachedis most often agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

3. Identification of Agonist Anti-trkC Antibodies

Agonist antibodies may be identified, for example, using the kinasereceptor activation (KIRA) assay described in U.S. Pat. Nos. 5,766,863and 5,891,650. This ELISA-type assay is suitable for qualitative orquantitative measurement of kinase activation by measuring theautophosphorylation of the kinase domain of a receptor protein tyrosinekinase (rPTK, e.g. trk receptor), as well as for identification andcharacterization of potential agonist or antagonists of a selected rPTK.The first stage of the assay involves phosphorylation of the kinasedomain of a kinase receptor, in the present case a trkC receptor,wherein the receptor is present in the cell membrane of a eukaryoticcell. The receptor may be an endogenous receptor or nucleic acidencoding the receptor, or a receptor construct, may be transformed intothe cell. Typically, a first solid phase (e.g., a well of a first assayplate) is coated with a substantially homogeneous population of suchcells (usually a mammalian cell line) so that the cells adhere to thesolid phase. Often, the cells are adherent and thereby adhere naturallyto the first solid phase. If a “receptor construct” is used, it usuallycomprises a fusion of a kinase receptor and a flag polypeptide. The flagpolypeptide is recognized by the capture agent, often a captureantibody, in the ELISA part of the assay. An analyte, such as acandidate agonist, is then added to the wells having the adherent cells,such that the tyrosine kinase receptor (e.g. trkC receptor) is exposedto (or contacted with) the analyte. This assay enables identification ofagonist ligands for the tyrosine kinase receptor of interest (e.g.trkC). It is also possible to use this assay to detect antagonists of atyrosine kinase receptor. In order to detect the presence of anantagonist ligand which blocks binding of an agonist to the receptor,the adhering cells are exposed to the suspected antagonist ligand first,and then to the agonist ligand, so that competitive inhibition ofreceptor binding and activation can be measured. Also, the assay canidentify an antagonist which binds to the agonist ligand and therebyreduces or eliminates its ability to bind to, and activate, the rPTK. Todetect such an antagonist, the suspected antagonist and the agonist forthe rPTK are incubated together and the adhering cells are then exposedto this mixture of ligands. Following exposure to the analyte, theadhering cells are solubilized using a lysis buffer (which has asolubilizing detergent therein) and gentle agitation, thereby releasingcell lysate which can be subjected to the ELISA part of the assaydirectly, without the need for concentration or clarification of thecell lysate.

The cell lysate thus prepared is then ready to be subjected to the ELISAstage of the assay. As a first step in the ELISA stage, a second solidphase (usually a well of an ELISA microtiter plate) is coated with acapture agent (often a capture antibody) which binds specifically to thetyrosine kinase receptor, or, in the case of a receptor construct, tothe flag polypeptide. Coating of the second solid phase is carried outso that the capture agent adheres to the second solid phase. The captureagent is generally a monoclonal antibody, but, as is described in theexamples herein, polyclonal antibodies may also be used. The cell lysateobtained is then exposed to, or contacted with, the adhering captureagent so that the receptor or receptor construct adheres to (or iscaptured in) the second solid phase. A washing step is then carried out,so as to remove unbound cell lysate, leaving the captured receptor orreceptor construct. The adhering or captured receptor or receptorconstruct is then exposed to, or contacted with, an anti-phosphotyrosineantibody which identifies phosphorylated tyrosine residues in thetyrosine kinase receptor. In the preferred embodiment, theanti-phosphotyrosine antibody is conjugated (directly or indirectly) toan enzyme which catalyses a color change of a non-radioactive colorreagent. Accordingly, phosphorylation of the receptor can be measured bya subsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.

Following initial identification, the agonist activity can be furtherconfirmed and refined by bioassays, known to test the targetedbiological activities. For example, the ability of anti-trkC monoclonalantibodies to mimic the activity of NT-3 can be tested in the PC12neurite outgrowth assay as described in Example 1, and confirmed inknown animal models of neurodegenerative diseases, such as theexperimental animal models of cisplatin- and pyridoxine-inducedneuropathies described in Example 2.

3. Therapeutic and Diagnostic Uses of Agonist Anti-TrkC Antibodies

The anti-trkC agonist antibodies of the present invention are believedto be useful in the treatment (including prevention) of disorders thepathology of which involves cellular degeneration or disfunction. Inparticular, the anti-trkC agonist antibodies are promising candidatesfor the treatment of various (chronic) neurodegenerative disorders andacute nerve cell injuries. Such neurodegenerative disorders include,without limitation, peripheral neuropathies; motorneuron disorders, suchas amylotrophic lateral schlerosis (ALS, Lou Gehrig's disease), Bell'spalsy, and various conditions involving spinal muscular atrophy orparalysis; and other human neurodegenerative diseases, such asAlzheimer's disease, Parkinson's disease, epilepsy, multiple schlerosis,Huntington's chorea, Down's Syndrome, nerve deafness, and Meniere'sdisease, and acute nerve cell injuries, for example due to trauma orspinal cord injury.

The anti-trkC antibodies of the present invention are believed to beparticularly suited for the treatment of peripheral neuropathy, aneurodegenerative disorder that affects the peripheral nerves, mostoften manifested as one or a combination of motor, sensory,sensorimotor, or autonomic dysfunction. Peripheral neuropathies may, forexample, be genetically acquired, can result from a systemic disease,can be induced by a toxic agent, such as a neurotoxic drug, e.g.antineoplastic agent, or industrial or environmental pollutant, or canbe idiopathic. Thus, peripheral sensory neuropathy is characterized bythe degeneration, decrease or failure of function of peripheral sensoryneurons, which may occur, for example, as a consequence of diabetes(diabetic neuropathy), cytostatic drug therapy in cancer (e.g. treatmentwith chemotherapeutic agents such as vincristine, cisplatin,methotrexate, or 3′-azido-3′-deoxythymidine), alcoholism, acquiredimmunodeficiency syndrome (AIDS), or genetic predisposition. Geneticallyacquired peripheral neuropathies include, for example, Refsum's disease,Krabbe's disease, Metachromatic leukodystrophy, Fabry's disease,Dejerine-Sottas syndrome, Abetalipoproteinemia, and Charcot-Marie-Tooth(CMT) Disease (also known as Proneal Muscular Atrophy or HereditaryMotor Sensory Neuropathy (HMSN)).

Based on the demonstrated ability of NT-3, the native ligand of the trkCreceptor, to promote proliferation of peripheral blood leukocytes, theanti-trkC agonist antibodies of the present invention may be used alsoas therapeutic agents for the treatment of neutropenia, variousinfections, and tumors. Since the expression of trkC is not limited toneurons, anti-trkC agonist antibodies are expected to find utility inthe prevention or treatment of disorders characterized by cellulardegeneration in general, without restriction to neural cells.

The anti-trkC antibodies of the present invention may also be used toinduce angiogenesis, or treat pathological conditions/diseases in whichthe induction of angiogenesis is desirable. Such pathological conditionsinclude, for example, cardiac ischemia regardless of the underlyingpathology, including cerebrovascular disorders caused by insufficientcerebral circulation. Angiogenesis may also be desirable in thetreatment of wounds, including ulcers, diabetic complications of sicklecell disease, and post surgical wounds.

The anti-trkC antibodies of the present invention may also be useful inthe diagnosis of diseases involving cellular degeneration, in particularthe neurodegenerative diseases listed above.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodycan be labeled with the radioisotope using the techniques described inCurrent Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed.Wiley-Interscience, New York, N.Y., Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) -D-galactosidase (-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl- -D-galactosidase) or fluorogenic substrate4-methylumbelliferyl- -D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g., digoxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the anti-trkC antibody need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the anti-trkC antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of trkC protein in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the cells or tissue ofinterest can be localized using immunoscintiography.

The antibodies may also be used as staining reagents in pathology,following techniques well known in the art.

The anti-trkC agonist antibodies of the present invention are believedto possess numerous advantages over NT-3 as therapeutic agents,including improved efficacy, improved pharmacokinetic properties (pK)and bioavailability, and lack of hyperalgesia following administration.

4. Pharmaceutical Formulations

Therapeutic formulations of the antibodies of the present invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

An effective amount of an antibody of the present invention to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 g/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer a molecule ofthe present invention until a dosage is reached that provides therequired biological effect. The progress of this therapy is easilymonitored by conventional assays.

Administration may be by any conventional route known in the artincluding, without limitiation, intravenous, subcutaneous, topical,intramuscular, intratracheal, intracerebral, intranasal, intrapulmonary,and intraparyncal administration.

4. Gene Therapy

The nucleic acid encoding the antibodies of the present invention mayalso be used in gene therapy of various (chronic) neurodegenerativedisorders and acute nerve cell injuries, especially genetically acquiredperipheral neuropathies. Two basic approaches to gene therapy haveevolved: ex viva gene therapy and in viva gene therapy. In ex vivo genetherapy, cells are removed from a subject and cultured in vitro. Afunctional replacement gene is introduced into the cells in vitro, themodified cells are expanded in culture, and then reimplanted in thesubject. In in vivo gene therapy, the target cells are not removed fromthe subject. Rather, the transferred gene is introduced into cells ofthe recipient in situ, that is, within the recipient.

Several ex vivo gene therapy studies in humans have been reported andare reviewed, for example, in Anderson, Science 256:808-813 (1992), andMiller, Nature 357:455-460 (1992).

The viability of in viva gene therapy has been demonstrated in severalanimal models, as reviewed in Felgner et al., Nature 349:351-352 (1991).Direct gene transfer has been reported, for example, into muscle tissue(Ferry et al., Proc Natl. Acad. Sci. 88:8377-8781 [1991]; Quantin etal., Proc. Natl. Acad. Sci. USA 89:2581-2584 [1992]); the arterial wall(Nabel et al., Science 244:1342-1344 [1989]); and the nervous system(Price et al., Proc. Natl. Acad. Sci. 84:156-160 [1987]).

Accordingly, the present invention also provides delivery vehiclessuitable for delivery of a polynucleotide encoding an agonist anti-trkCantibody into cells (whether in vivo or ex vivo). Generally, apolynucleotide encoding an antibody (e.g. linear antibody or antibodychains) will be operably linked to a promoter and a heterologouspolynucleotide. Delivery vehicles suitable for incorporation of apolynucleotide encoding an antibody of the present invention forintroduction into a host cell include non-viral vehicles and viralvectors. Verma and Somia, Nature 389:239-242 (1997).

A wide variety of non-viral vehicles for delivery of a polynucleotideencoding an antibody of the present invention are known in the art andare encompassed in the present invention. A polynucleotide encoding ananti-trkC antibody can be delivered to a cell as naked DNA (U.S. Pat.No. 5,692,622; WO 97/40163). Alternatively, a polynucleotide encoding ananti-trkC antibody herein can be delivered to a cell associated in avariety of ways with a variety of substances (forms of delivery)including, but not limited to cationic lipids; biocompatible polymers,including natural polymers and synthetic polymers; lipoproteins;polypeptides; polysaccharides; lipopolysaccharides; artificial viralenvelopes; metal particles; and bacteria. A delivery vehicle can be amicroparticle. Mixtures or conjugates of these various substances canalso be used as delivery vehicles. A polynucleotide encoding an antibodyherein can be associated non-covalently or covalently with these variousforms of delivery. Liposomes can be targeted to a particular cell type,e.g., to a glomerular epithelial cell.

Viral vectors include, but are not limited to, DNA viral vectors such asthose based on adenoviruses, herpes simplex virus, poxviruses such asvaccinia virus, and parvoviruses, including adeno-associated virus; andRNA viral vectors, including, but not limited to, the retroviralvectors. Retroviral vectors include murine leukemia virus, andlentiviruses such as human immunodeficiency virus. Naldini et al.,Science 272:263-267 (1996).

Non-viral delivery vehicles comprising a polynucleotide encoding ananti-trkC antibody can be introduced into host cells and/or target cellsby any method known in the art, such as transfection by the calciumphosphate coprecipitation technique; electroporation;electropermeabilization; liposome-mediated transfection; ballistictransfection; biolistic processes including microparticle bombardment,jet injection, and needle and syringe injection; or by microinjection.Numerous methods of transfection are known to the skilled worker in thefield.

Viral delivery vehicles can be introduced into cells by infection.Alternatively, viral vehicles can be incorporated into any of thenon-viral delivery vehicles described above for delivery into cells. Forexample, viral vectors can be mixed with cationic lipids (Hodgson andSolaiman, Nature Biotechnol. 14:339-342 [1996]); or Iamellar liposomes(Wilson et al. Proc. Natl. Acad. Sci. USA 74:3471 [1977]; and Faller etal J. Virol. 49:269 [1984]).

In a preferred embodiment, nucleic acid encoding both the heavy and thelight chains (including fragments) of an anti-trkC antibody of thepresent invention will be present in the same polycistronic expressionvector, such as those disclosed in U.S. Pat. Nos. 4,965,196 and4,713,339. Polycistronic expression vectors contain sequences coding fora secondary protein and a desired protein, wherein both the desired andsecondary sequences are governed by the same promoter. The codingsequences are separated by translational stop and start signal codons.The expression of the secondary sequence effects control over theexpression of the sequence for the desired protein, and the secondaryprotein functions as a marker for selection of transfected cells.

In in vivo gene therapy, the vector may be administered to therecipient, for example, by intravenous (i.v.) injection. Suitable titerswill depend on a variety of factors, such as the particular vectorchosen, the host, strength of promoter used, and the severity of thedisease being treated.

The invention will be further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Production and Characterization of Agonist Anti-trkCMonoclonal Antibodies

Production and Isotyping of Antibodies

Wild type Balb/C mice and transgenic mice producing human IgG2 or IgG4(Xenomice, described in Mendez et al., Nature Genetics 15: 146-156[1997]) were hyperimmunized either intraperitoneally, via rear footpad,or subcutaneously with 20 μg of human trkC-IgG (Shelton et al., J.Neurosci. 15: 477-491 [1995]) in either Frieund's or Ribi adjuvant asdescribed in Mendez et al. (supra). Spleen cells from the immune micewere fused with myeloma cells (X63.Ag8.653, ATCC Rockville, Md.). Atotal of 33 fusions were performed using 253 Xenomice and 35 wild typeBalb/C mice. Plates (21,734 wells total) were initially screened bydirect ELISA using trkC-IgG. The ELISA screen identified 684 trkCpositive hybridomas, all of which were then evaluated for agonistactivity in trkC KIRA (Kinase activated Receptor Assay). The KIRAidentified 14 Xenomouse derived and 22 wild type Balb/C mice derivedhybridomas secreting anti-trkC agonist antibodies. These hybridomas weresubcloned by limiting dilution, reassayed to confirm agonist activity,and were used to induce ascites by injecting into Pristane-primed Balb/Cor nude mice (Hongo et al., Hybridoma 14: 253-260 [1995]). Themonoclonal antibodies present in ascites were purified by Protein Aaffinity chromatography (Hongo et al., supra). Specific fusionefficiency (number of positives/number of wells screened) was 3% forboth the Xenomouse and wild type Balb/C mouse fusions. The incidence ofagonist monoclonal antibodies (agonists/number of trkC ELISA positives)was 3% and 8% for the Xenomouse and wild type Balb/C mouse fusions,respectively. Isotypes of the murine monoclonal antibodies weredetermined using either GIBCO BRL dipstick or Zymed mouse-typerisotyping kit, following supplier's instructions. The Xenomice wereeither IgG₂ or IgG₄ strain, producing corresponding isotypes ofantibodies. Table 1 shows isotypes of various human and murine anti-trkCmonoclonal antibodies. A total of 8 human IgG₂, 6 human IgG₄, 7 murineIgG₁, 10 murine IgG₂, and 5 murine IgG_(2b) monoclonal antibodies wereidentified. The monoclonal antibodies with the most potent agonistactivity (depicted by asterisk in Table 2), as determined by KIRA assay,were selected for in-depth characterization.

TABLE 2 Human Mabs (14 Total) IgG₂ Isotype (8 Mabs) IgG₄ Isotype (6Mabs) 2.5.1*    4.8 6.1.2* 2337 6.4.1* 2338 2342 2339 2343 2348  2344* 2349*  2345* 2346 Murine Mabs (22 Total) IgG₁ IgG_(2a) IgG_(2b) (7)(10) (5) IgG₃ 2249  2248* 2252  2250* 2272 2273  2253* 2251 2277 22542255 2279  2256* 2274 2280 2257 2275 2260 2276 2278 2281 2282

Determination of Agonist Activity

a. KIRA Assay

Two bioassays were used to determine NT-3 agonist activity of anti-trkCmonoclonal antibodies. The Kinase activated receptor assay (KIRA), whichhas been discussed in greater detail hereinabove, measures tyrosinephosphorylation of trkC in transfected cells in response to stimulationwith a ligand, such as NT-3, or agonist monoclonal antibodies (Sadick etal., Exp. Cell Res. 234: 354-361 [1997]). The monoclonal antibodies werediluted to 27 μg/ml in KIRA stimulation buffer (F12/DMEM 50:50containing 2% bovine serum albumin (BSA; Intergen Co., Purchase, N.Y.)and 25 mM Hepes, 0.2 μm filtered). The monoclonal antibodies werefurther diluted serially 1:3 (8 dilutions total; concentrations rangingfrom 0.01-180 nM) in stimulation medium. Chinese Hamster Ovary (CHO)cells stably transfected with trkC fused with a 26 amino acidpolypeptide flag epitope derived from HSV glycoprotein D (gD) wereseeded (5×10⁴ cells/well) and grown in 96-well cell culture plates. Thecells were then stimulated with either NT-3 (as a positive control) orvarious anti-trkC monoclonal antibodies, using serial dilutions of 0.1;1.56; 3.13; 6.25; 12.5; 25; 50 and 100 ng/ml. All dilutions were assayedin duplicate for 6 hours. The assay was carried out essentially asdescribed in Sadick et al. (supra). Briefly, cells were lysed usingTriton X-100 and trkC present in lysate captured in ELISA usingantibodies against the gD epitope and phosphorylated trkC detected andquantitated using anti-phosphotyrosine antibodies suitably conjugatedwith enzyme. A monoclonal antibody not directed against trkC (anti-IL8IgG₂ Xenomous-derived human antibody or anti-gp120 IgG₁ murinemonoclonal antibody) was used as a negative control. As shown in FIG. 1(A and B), all the selected anti-trkC monoclonal antibodies could mimicthe activity of NT-3 inasmuch as they could stimulate tyrosinephosphorylation of trkC receptor. The human anti-trkC monoclonalantibodies (FIG. 1A) showed more potent agonistic activity than themurine anti-trkC monoclonal antibodies (FIG. 1B). For example, the besthuman anti-trkC monoclonal antibody is 10-fold more potent than the bestmurine anti-trkC monoclonal antibody. Furthermore, the human monoclonalswere nearly as efficient as NT-3 especially in the lower range ofconcentration.

b. PC12 Neurite Outgrowth Assay

Another assay used to determine NT-3 mimetic activity of anti-trkCmonoclonal antibodies was PC12 neurite outgrowth assay. This assaymeasures the outgrowth of neurite processes by rat pheocytochroma cells(PC12) in response to stimulation by appropriate ligands. These cellsexpress endogenous trkA and are therefore responsive to NGF. However,they do not express endogenous trkC and are therefore transfected withtrkC expression construct in order to elicit response to NT-3 and itsagonists. PC12 cells were transfected (Urfer et al., Biochem.36:4775-4781 [1997]; Tsoulfas et al., Neuron 10:975-990 [1993]) withfull-length human trkC and plated in 96-well cell culture plates (1000cells/well). Three days following transfection, anti-trkC monoclonalantibodies were added in triplicate (concentration ranging from 0.0002to 2.7 nM) and incubated for an additional 3 days at 37° C. The cellswere then analyzed by phase contrast microscopy and cells with neuritesexceeding 2 times the diameter of the cell were counted. The human aswell as the murine anti-trkC monoclonal antibodies could stimulateneurite outgrowth in PC12 cells as shown in FIGS. 1C and D. The humananti-trkC monoclonal antibodies (FIG. 1C) exhibited far more potentactivity than the murine anti-trkC monoclonal antibodies (FIG. 1D) thuscorroborating the results obtained in the KIRA assay. Furthermore,consistent with the KIRA assay results, the human anti-trkC monoclonalantibodies showed roughly similar stimulation as obtained with NT-3. Theresults obtained with the two bioassays described above demonstrate theability of anti-trkC monoclonal antibodies to mimic the activity ofNT-3, the natural ligand of trkC receptor.

Agonist activity of the monoclonal antibodies was ranked according tomaximum induction of tyrosine phosphorylation and calculated EC50 of thephosphorylation curves in the KIRA assay and PC12 neurite outgrowthassay. Table 3 summarizes characteristics of various anti-trkC agonistmonoclonal antibodies.

TABLE 3 Agonist Immunoblot Activity Binds NR/Red. Affinity MAb IDIsotype KIRA/PC12 Rat trkC NR Red. Kd (nM) Human MAbs 2.5.1 G2 (+++/+++)NO ++ ++ 12 6.1.2 G2 (++++/++++) NO + + 12.5 6.4.1 G2 (++++/++++)YES + + 12 2344 G2 (+++/+++) NO ++ + 19 2345 G2 (++++/++++) NO ++ ++12.1 2349 G4 (++++/++++) NO ++ + 23 Murine MAbs 2248 G2a (+/+) NO +++ −5.9 2250 G1 (++/++) NO ++ ++ 8.7 2253 G1 (++/+) NO ++ ++ 42 2256 G1(+/+) YES ++ + 62

Testing Specificity of Anti-trkC Antibodies

The specificity of anti-trkC monoclonal antibodies was tested usingdirect ELISA. The microtiter plates were coated overnight withimmunoadhesin construct of the receptor trkA-IgG, trkB-IgG or trkC-IgGas capture antigens (described in Shelton et al., J. Neurosci. 15:477-491 [1995]) using 100 μl of 1 μg/ml solution diluted in 50 mMcarbonate buffer, pH 9.5. CD4-IgG (Capon et al., Nature 337: 525-531[1989]) was used in place of capture antigen as a negative control. Thecoated plates were incubated for 1 hr at room temperature with variousconcentration of anti-trkC monoclonal antibodies (100 μl of 0.01 to 1μg/ml) diluted in PBS containing 0.5% BSA and 0.05% Tween 20. Afterwashing to remove excess unbound antibodies, appropriate HRP conjugate(human monoclonal antibodies: goat anti-human K-HRP, 1:5000 diluted;murine monoclonal antibodies: goat anti-mouse IgG (Fc)-HRP, 1:5000diluted) was added and incubated for 1 hr at room temperature. Theplates were then washed, developed and read as previously described(Hongo et al., Hybridoma 14: 253-260 [1995]). FIG. 2 shows arepresentative example using a human anti-trkC monoclonal antibody6.1.2. The binding was highly specific to trkC, and no significantcross-reaction was observed with either trkA or trkB. Similarly, otherhuman and mouse anti-trkC monoclonal antibodies showed specificrecognition of trkC.

The binding of various anti-trkC agonist monoclonal antibodies to humantrkC and rat trkC was compared using a direct ELISA essentially asdescribed above except the capture antigen used for human trkC wastrkC-gD instead of trkC-IgG. Results shown in FIG. 3 indicate that amonghuman anti-trkC monoclonal antibodies, only 6.4.1 significantlyrecognized rat trkC, rest were specific for human trkC. Similarly, amongmurine monoclonal antibodies, only 2256 recognized rat trkC to asignificant extent while others showed specific recognition of humantrkC only.

Affinity Studies

Affinities of anti-trkC agonist monoclonal antibodies were determinedusing using BIAcore-2000™ surface plasmon resonance (SPR) system(BIAcore, Inc., Piscataway, N.J.). CM5 biosensor chips were activatedwith N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)and N-hydroxysuccinimide (NHS) according to the supplier's instructions.In the first series of binding experiments, the antigen, gD-tagged trkC,was diluted into 10 mM sodium acetate buffer (pH 4.8), and injected overthe activated chip at a concentration of 0.09 mg/mL. Using variableexposure times, four ranges of antigen density were achieved:14,000-17,000 response units (RU), 7000-9000 RU, 2000-3000 RU, and400-600 RU. The chip was blocked with ethanolamine.

In the first series of kinetic measurements, anti-trkC antibodies(IgG's) were diluted into running buffer (PBS containing 0.05% Tween-20and 0.01% sodium azide) and 0.03 mL (667 nM) was injected over thebiosensor chip at 25° C. at a flow rate of 0.01 mL/min. Regeneration wasachieved with a 30 sec pulse of 10 mM HCl, followed by a 1 min pulse of100 mM Tris-HCl pH 8.0 and two wash steps.

In a second series of experiments, the IgG's (0.1 mg/mL in 10 mM sodiumacetate, pH 4.8) were immobilized as described above, except thatantibody density was limited to 1000-2000 RU. Two-fold serial dilutionsof gD-trkC in the range of 3.7 M to 29 nM were then injected over thebiosensor chip for kinetics measurements as described above.

The dissociation phase of each kinetic curve were fit to a singleexponential dissociation rate (k_(off)), and these rates were used inthe calculation of the association rate (k_(on)) from the injectionphase, using a simple 1:1 Langmuir binding model (Lofas & Johnsson,1990).

Equilibrium dissociation constants, K_(d)'s, from SPR measurements werecalculated as the ratio k_(off)/k_(on).

The affinities of anti-trkC antibodies for gD-trkC were measured in SPRkinetics experiments with either antigen or antibody immobilized.Apparent affinities determined from experiments using low densities ofimmobilized antigen (0.4 to 0.6 ng/mm2), were generally consistent withthose determined in experiments using immobilized IgG (see Table 2).However, at higher densities of immobilized gD-trkC, the apparentbinding affinity of each antibody became progressively tighter byfactors of as much as 10 fold, probably because of an avidity effect ofbinding by the bivalent IgG (data not shown). In some cases, no bindingcould be detected when trkC was injected over immobilized IgG. This mayhave occurred because immobilization of the IgG led to steric blockingof the antigen-binding site. Under all conditions tested, the antibody2248 had the highest apparent affinity (K_(d)=5.6 to 8.5 nM) of allantibodies tested.

Binding affinities determined by SPR. Results are shown for IgG'sbinding to immobilized gD-trkC (400-600 RU) and for gD-trkC binding toimmobilized IgG's (1000-3000 RU). K_(d) (nM) Antibody (IgG) ImmobilizedgD-trkC Immobilized IgG 2248 5.9 8.5 2250 8.7 28 2253 42 51 2256 62 3002344 19 NDB 2345 12 NDB 2349 23 NDB 6.4.1 12 28 6.1.2 13 16 2.5.1 12 NDBNDB = no detectable binding.

Competition ELISA

A competition ELISA was used to get preliminary information aboutvarious groups to which these antibodies belong depending on theepitope(s) on trkC they recognize. In this assay, trkC-gD (1 μg/ml) wasused as a capture antigen to coat microtiter plate. A specificbiotinylated anti-trkC monoclonal antibody (1 μg/ml) was added to thecoated plate either alone or in presence of another anti-trkC monoclonalantibody that was unlabeled and used in excess (50 μg/ml) as compared tothe labeled antibody. If biotinylated antibody and unlabeled antibodyboth recognize the same or overlapping epitope, they will compete forbinding to the immobilized trkC, resulting in decreased binding of thelabeled antibody. If they recognize different and non-overlappingepitopes, there will be no competition between them, and the binding ofthe labeled antibody to the immobilized trkC will not be affected.Unlabeled human IgG2 and mouse IgG were used as negative control. Arepresentative data in FIG. 4 shows that all anti-trkC monoclonalantibodies, except murine anti-trkC 2248 monoclonal antibody, competewith labeled human anti-trkC 6.1.2 monoclonal antibody for binding tothe immobilized trkC, suggesting that murine 2248 antibody recognizes anepitope on trkC that is different from the epitope(s) recognized by allother anti-trkC antibodies.

It is interesting to note that when unlabeled murine monoclonal antibody2248 is bound first to immobilized trkC, none of the other(biotinylated) antibodies can access their binding site, suggeting thateven though the epitopes are distinct, steric hinderance may play arole. Such pairwise comparison gives valuable information and helps inclassifying antibodies directed against the same antigen into differentgroups based on epitope recognition. A summary of such comparison isshown in FIG. 5. The results indicate that the antibodies can be dividedinto two distinct groups: Group 1 encompasses all monoclonal antibodiesexcept 2248, whereas Group 2 is composed of 2248.

Epitope Mapping with Domain Swap Mutants

Further epitope mapping was performed utilizing chimeric trkC in whichvarious domains were replaced with corresponding domains from trkA ortrkB. This approach was made possible by the fact that anti-trkCantibodies do not significantly cross-react with trkA or trkB. The useof such domain-swap mutants has a distinct advantage over deletionmutants. The deletion of a domain might disrupt the secondary structureof protein whereas substitution of a domain with a corresponding domain,of similar size and substantially similar amino acid sequence, from arelated protein in domain-swap mutants is likely to retain the secondarystructure. The extracellular domain of trk receptors is composed of 5domains as shown in FIG. 6A. D1 and D3 are cysteine-rich domains, D2 isa leucine-rich domain, and D4 and D5 are immunoglobulin-like domains.Domain-swap mutants of trkC containing replacement of D1, D4 and D5 withthe corresponding domains from trkB or trkA were made (Urfer et al.,EMBO J. 14:2795-2805 [1995]). Wild type trkC and wild type trkA wereused as positive and negative controls respectively. The domain-swapmutants of trkC are designated according to the source of the replaceddomain. For example, s1B has D1 domain from trkB, s4B has D4 domain fromtrkB, s5B has D5 domain from trkB, and s5A has D5 domain from trkA. Allof the mutants were expressed as immunoadhesin, i.e. fused to IgG, andpurified.

The binding of each of the agonist anti-trkC monoclonal antibody tovarious domain-swap mutants was evaluated by ELISA. F(ab′)₂ fragmentfrom goat anti-human IgG was used for coating microtiter plates tocapture serial dilutions (100 μg/ml to 2.4 ng/ml, 100 μl/well, one hourat room temperature) of immunoadhesins (trkC-IgG, trkA-IgG anddomain-swap mutants of trkC as immunoadhesin). Either unlabeled human orbiotinylated murine anti-trkC monoclonal antibodies were added (100 μlper well; 1 μg/ml, one hour at room temperature) to the platescontaining immobilized immunoadhesins, washed to remove unbound excessreagents, and incubated with goat anti-human κ or streptavidinconjugated to HRP. As shown in FIG. 6B, all human anti-trkC monoclonalantibodies were able to recognize trkC domain-swap mutants withreplacement of domain D1 or D4. However, replacement of domain D5 withthe corresponding domain derived from either trkB or trkA destroyedrecognition by anti-trkC antibodies. The extent of binding was reducedto the same low level as that observed with a negative control, trkA.The results suggest that all the human anti-trkC monoclonal antibodiestested recognize an epitope located somewhere in domain D5.

Similar analysis was performed with murine anti-trkC monoclonalantibodies essentially the same way except the secondary antibody usedwas goat anti-mouse IgG Fc coupled to HRP. As with the human anti-trkCantibodies, the replacement of domain D5 abolished binding to all themurine anti-trkC monoclonal antibodies tested (FIG. 6B). Additionally,the replacement of domain D4 also destroyed the binding of 2248 murineanti-trkC antibody. The human as well as murine anti-trkC agonistmonoclonal antibodies all seem to recognize an epitope in domain 5 withthe exception of 2248 murine antibody, which seems to additionallyrecognize a determinant in domain 4. It appears that 2248 epitope may bea linear epitope overlapping the boundary of domain 4 and 5.Alternatively, 2248 antibody might recognize a secondary structureformed by discontiguous epitope with determinants derived from bothdomain 4 and domain 5. Interestingly, Urfer et al. (J. Biol. Chem. 273:5829-5840 [1998]) have earlier established the prominent role of domain5 in trkC receptor for mediating the interaction with NT-3.Surprisingly, the antibodies described herein also bind to an epitope oftrkC which is largely overlapping with that recognized by NT-3. This issurprising because of the relative sizes and shapes of NT-3 andimmunoglobulin molecules. The likely mode of action of these activatorsis to crosslink the extracellular domains of two trkC molecules in sucha way to bring together their intracellular tyrosine kinase domains andcross phosphorylate and activate them.

In homodimeric NT-3, it has been established that the two areas of themolecule which interact with trkC are diametrically opposed on oppositesides of the molecule, 180 degrees apart from each other. The distancebetween these areas is on the order of 16 Å. On the other hand, the twotrkC interacting sites in the immunoglobulin molecules described hereare not diametrically opposed. In addition to displaying the trkCbinding domains at a different angle than NT-3, immunoglobulins willhave the trkC binding domains separated from each other by a much widerdistance than they are in NT-3. This will vary with the exact angle ofthe two Fab domains, but is in the range of 50 Å to 150 Å. It would havebeen difficult to have foreseen that two such very differentcrosslinkers as NT-3 and the agonist Mabs act as agonists when bound tothe same site on trkC.

Site-Directed Mutagenesis

Site-directed mutagenesis approach was used to determine thecontribution of selected individual amino acid residues of domain 5 inthe recognition by anti-trkC antibodies. FIG. 7 shows the amino acidsequence of human trkC domains 4 and 5. All dotted residues weremutagenized to alanine except residues L284, L286 and E287 which werechanged to E, H, and K respectively (Urfer et al., J. Biol. Chem. 273:5829-5840 [1998]). A total of 26 single amino acid mutations were madeand evaluated for their effect on binding to anti-trkC monoclonalantibodies. The values shown in Table 5 represent the ratio of bindingto anti-trkC antibody of mutant vs wildtype trkC. In order to minimizevariation and provide effective comparison, EC50 values were determinedfor each mutant for each antibody and divided by the EC50 value obtainedwith wildtype trkC.

TABLE 5 trkC Mutant NT-3* 2.5.1 6.1.2 6.4.1 2344 2345 2349 2248 22502253 2256 1436 trkC 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0R275A 1.1

E280A 0.7

E283A 1.7

L284E 1.1 NB NB NB NB NB NB 0.7 0.6 0.7 1.1 0.8 R285A 1.5 1.1 1.2 0.80.9 1.0 0.9 0.8 2.6 0.9 NB 0.4 L286H 1.2 0.6 1.3? NB 0.9 0.5 0.6 0.6 0.40.4 0.6 0.6 E287K 27.3 NB NB NB NB NB NB 0.6 0.8 0.7 0.7 0.6 E291A 1.0

R295A 11.6

Q309A 1.0

R312A 0.8

K315A 0.9

H318A 1.0 0.8 1.1 0.7 0.8 1.0 0.7 1.0 1.0 0.8 0.8 1 E320A 1.0

E324A 1.2

E329A 1.0

N335A 37.8 NB NB 0.3 NB NB 1.5 0.6 0.5 0.5 0.5 0.6 K336A 0.9

T338A 30.3

H339A 1.7

K350A 1.0

Q358A 1.2

K366A 0.9

E367A 1.2

D372A 1.2

E373A 1.1

The gray areas indicate that the designated mutants did not have aninitial effect on monoclonal antibody binding, and were therefore notre-assayed. Mutations that completely obliterated monoclonal antibodybinding are shown as NB (“no binding observed”). The analysis indicatesthe major contribution of amino acid residues L284, E287 and N335 oftrkC in recognition by anti-trkC agonist monoclonal antibodies tested. Amodel of the complex of trkC domain 5 with NT3 shows the position ofthese residues in close contact with CDRs of antibody (FIG. 8). Thismodel is based on the crustal structure of the complex of trkA domain 5wih NGF. For further details see, e.g. Urfer et al. J. Biol. Chem.(1998), supra, or Ultsch et al., J. Mol. Biol. 290:149-159 (1999).

Cloning and Sequencing of Antibody Variable Regions

In order to better understand the molecular basis of interaction betweentrkC and anti-trkC monoclonal antibodies, the heavy and light chainvariable sequences of agonist antibodies were cloned and DNA sequencedetermined. Total RNA was isolated from hybridoma cells producing thehuman and murine anti-trkC antibodies using RNA isolation kit fromStratagene (La Jolla, Calif.). RNA was reverse transcribed into cDNAusing SuperScript II system (Life Technologies, Inc., Gaithersburg, Md.)and specific 3′ primers based on framework 4 sequences derived from therespective heavy or light chain subgroup (Kabat and Wu, J. Immunol 147:1709-1719 [1991]). Subsequent PCR amplification was performed usingAmpliTaq DNA polymerase (Perkin Elmer, Foster City, Calif.) in presenceof 2.5 M DMSO with specific forward primers based on the N-terminalamino acid sequences of heavy and light chains and the same 3′ primersused for cDNA synthesis. PCR products were subcloned into an F(ab)′₂vector containing both human heavy and light chain constant regions(Carter et al., Bio/Technology 10: 163-167 [1992]). Five clones each ofthe V_(H) and V_(L) domains were sequenced and a consensus sequence wasobtained.

FIG. 9 shows the deduced amino acid sequences of heavy chain ofanti-trkC agonist monoclonal antibodies (2250, SEQ ID NO: 42; 2253, SEQID NO: 43; 2256, SEQ ID NO: 44; 6.1.2, SEQ ID NO: 45; 6.4.1, SEQ ID NO:46; 2345, SEQ ID NO: 47; and 2349, SEQ ID NO: 48). The deduced aminoacid sequences of light chain of anti-trkC agonist monoclonal antibodiesare shown in FIG. 10 (2250, SEQ ID NO: 49; 2253, SEQ ID NO: 50; 2256,SEQ ID NO: 51; 6.1.2, SEQ ID NO: 53; 6.4.1, SEQ ID NO: 53; 2345, SEQ IDNO: 54; and 2349, SEQ ID NO: 55). In both FIG. 9 and FIG. 10 theComplementarity Determining Regions (CDRs) are labeled as CDR1, CDR2 andCDR3, and the corresponding amino acid residues are shown in bold face.FIG. 11 summarizes the sequences of CDRs of heavy chain as well as lightchain of various anti-trkC monoclonal antibodies along with designationof respective heavy and light chain variable family to which theybelong.

Based on the determined amino acid sequences of the CDRs of the heavyand light chains of the anti-trkC agonist monoclonal antibodies, it ispossible to provide a general formula for several of these regions. Forthe murine antibodies, the heavy chain CDR1 may be represented by theformula XaaWXaaXaaWVK (SEQ ID NO:37), wherein Xaa at position 1 is F orY, Xaa at position 3 is I or M and Xaa at position 4 is E or H. Themurine heavy chain CDR2 may be represented by the formulaEIXaaPXaaXaaXaaXaaTNYNEKFKXaa (SEQ ID NO:38), wherein Xaa at position 3is L or Y, Xaa at position 5 is G or S, Xaa at position 6 is S or N, Xaaat position 7 is D or G, Xaa at position 8 is N or R and Xaa at position17 is G or S. The murine heavy chain CDR3 may be represented by theformula KNRNYYGNYVV (SEQ ID NO:12) or KYYYGNSYRSWYFDV (SEQ ID NO:13).For the human antibodies, the heavy chain CDR1 may be represented by theformula XaaXaaXaaYYWXaa (SEQ ID NO:39), wherein Xaa at position 1 is Sor 1, Xaa at position 2 is G or S and Xaa at position 3 is G, T or Y andXaa at position 7 is S or N. The human heavy chain CDR2 may berepresented by the formula XaaIXaaXaaSGSXaaTXaaNPSLKS (SEQ ID NO:40),wherein Xaa at position 1 is Y or R, Xaa at position 3 is Y or F, Xaa atposition 4 is Y or T, Xaa at position 8 is S or R and Xaa at position 10is N or Y. The human heavy chain CDR3 may be represented byDRDYDSTGDYYSYYGMDV (SEQ ID NO:14), DGGYSNPFD (SEQ ID NO:15) or theformula ERIAAAGXaaDYYYNGLXaaV (SEQ ID NO:41) wherein Xaa at position 8is A or T and Xaa at position 16 is D or A.

The deduced amino acid sequence of heavy and light chain variableregions was confirmed by determination of N-terminal peptide sequence ofthese antibodies. Electroblotting onto Millipore Immobilon-PSQ membraneswas carried out for 1 hr at 250 mA constant current in a BioRadTrans-Blot transfer cell (Matsudaira, J. Biol. Chem. 262: 10035-10038[1987]). The PVDF membrane was stained with 0.1% Coomassie Blue R-250 in50% methanol, 0.5 min. and destained for 2-3 min. with 10% acetic acidin 50% methanol. The mebrane was thoroughly washed with water andallowed to dry before storage at 20° C. Automated protein sequencing wasperformed on model 494A Perkin-Elmer sequencer (Perkin-ElmerCorporation, Foster City, Calif.) equipped with on-line PTH analyzer.Protein electroblotted onto PVDF membrane were sequenced in 6 mm microglass cartridge. Peaks were integrated with Justice Innovation softwareusing Nelson Analytical 760 interfaces. Sequence interpretation wasperformed on a DEC Alpha (Henzel et al., J. Chromatography 404: 41-52[1987]). Table 6 summarizes the classification of human and murineanti-trkC agonist monoclonal antibodies based on their N-terminalsequences.

TABLE 6 Heavy chain Light chain Human anti-trkC agonist mAbs 6.1.2Subgroup II Kappa I 6.4.1 Subgroup II Kappa I 2345 Subgroup II Kappa III2349 Subgroup II Kappa III 2.5.1 Subgroup II Kappa I 2344 Subgroup IIKappa I Murine anti-trkC agonist mAbs 2248 Subgroup IIA Kappa I 2250Subgroup IIA Kappa I 2253 Subgroup IIA Kappa IV 2256 Subgroup IIA KappaIII

Example 2 Effect of Agonist Anti-trkC Monoclonal Antibodies onNeuropathies in Experimental Animal Model

The principal use of NT-3 agonists is in the treatment and/or preventionof peripheral neuropathies. It is known that large fiber myelinatedsensory neurons, which are involved in mediating proprioception andvibration sense, express trkC that acts as a high affinity receptor forNT-3. Neuropathies involving these large fibers are common in diabetesand are also induced in response to certain chemotherapeutic agentsparticularly cisplatin and pyridoxine. NT-3 has shown efficacy in animalmodels of experimental diabetic neuropathy and cisplatin inducedneuropathy. However, the use of NT-3 is severely hampered by its poorbioavailability as shown in a rodent model. The use of anti-trkCMonoclonal antibodies as agonist of NT-3 offers numerous advantages andobviates a number of potential problems associated with the use of NT-3.

The in vivo half-life of agonist anti-trk monoclonal antibodies wasdetermined by injecting either intravenously or subcutaneously inexperimental animals. Shown on FIG. 12 are serum levels of monoclonalantibody 2256 at various times after intravenous (IV) injection of 1mg/kg or subcutaneous (subQ) injection of 5 mg/kg in rats. The serumlevels were determined b using the KIRA assay to measure the amount offully functional antibody 2256 by its ability to increase tyrosineautophosphorylation of trkC. These data indicate that monoclonalantibody 2256 in the rat has a half-life of 9 days and a bioavailabilityof 69% after subcutaneous administration. These values are consistentwith those obtained with other antibodies, and are distinctly differentfrom those obtained with NT3. Also shown in FIG. 12 is data obtainedafter injection of NT-3 at the same doses and routes as shown for Mab2256 (1 mg/kg, IV; 5 mg/kg subQ). These data indicate a serum half-lifeon the order of 4-5 minutes for NT-3, and a subcutaneous bioavailabilityof 7%. These data indicate that the antibodies are a significantimprovement over NT-3 in terms of the very important properties ofbioavailability and in vivo serum half-life.

It has been shown in two animal models of large fiber sensory neuropathythat NT-3 can protect or reverse the effects of chemical insult. Veryhigh doses of NT3 have been shown to protect large fiber sensory neuronsfrom the toxic effects of high doses of pyridoxine, and more moderatedoses of NT3 have been shown to reverse the effects of cisplatinumadministration. Since there might be many differences in the tissuedistribution of NT-3 and the agonist Mabs described here, it isimportant to determine whether the in vitro activity of the Mabstranslates into efficacy in animal models.

In order to create an animal model of cisplatinum induced neuropathy,adult rats were dosed with cisplatinum twice a week for sixteen weekswith 1 mg/kg intraperitoneally (IP). At this point, rats were split intofour groups. All four groups continued receiving cisplatinum twiceweekly. In addition to the continued cisplatinum, one group receivedNT-3 at a dose of 1 mg/kg, three times per week, one group received Mab2256 at a dose of 1 mg/kg once a week, one group received Mab 6.4.1 at adose of 1 mg/kg once a week, and one group received saline three times aweek. The NT-3 doses were given subcutaneously, while the Mabs andsaline were administered IV. This treatment regime was continued for anadditional four weeks, for a total of twenty weeks of cisplatinumadministration.

The function of large fiber sensory neurons was assessed in theseanimals electrophysiologically, by use of H-wave recording (Gao et al.,Ann. Neurol. 38(1):30-7 [1995]) As can be seen from the data shown inFIG. 13, the sensory conducton velocity was very low in the animalstreated with cisplatinum with saline alone. NT-3 treatment three times aweek caused an improvement of this lowered conduction velocity, as didtreatment with either Mab 2256 or Mab 6.4.1 once a week. The magnitudeof the improvement seen with the monoclonal antibodies used once a weekwas at least as great as that seen with three times a week treament withNT-3.

Pyridoxine is also known to induce a sensory neuropathy that primarilydamages the large myelinated subpopulation of sensory neurons (Helgrenet al., J. Neurosci. 17(1):372-82 [1997]). High doses of NT3 have beenshown to block the development of this neuropathy (Helgren et al.,supra). Treatment of animals with two different doses of pyridoxine(either 400 mg/kg or 600 mg/kg daily, IP) for two weeks causes damage tothe large neurons of the DRG. This damage can be detected by a decreasein the expression of several proteins known to be expressed eitherpreferentially or exclusively by large neurons in the ORG. Theexpression level of these markers was assessed by measuring the level ofthe mRNA encoding them by use of the TAQMAN RT-PCR technique.

Taqman RT-PCR for trkC Agonist Effects:

A. Probes and Primers NFL F-CAGCAGAACAAGGTCCTGGAA 21MER (SEQ ID NO: 72)R-AGCGGGAAGGCTCTGAGTG 19MER (SEQ ID NO: 73) P-AGCTGTTGGTGCTGCGCCAGAA22MER (SEQ ID NO: 74) NSE F-TCCATTGAAGACCCATTCGAC 21MER (SEQ ID NO: 75)R-GCCGACATTGGCTGTGAAC 19MER (SEQ ID NO: 76) P-AGGATGACTGGGCAGCTTGGTCCA24MER (SEQ ID NO: 77) TRKC F-CAGCCCACTGCACCATATCA 20MER (SEQ ID NO: 78)R-CTGTATCCGGCCCAGCAT 18MER (SEQ ID NO: 79) P-CCATGGCATCACTACACCTTCATCGCT27MER (SEQ ID NO: 80) CALRET F-TGGGAAAATTGAGATGGCAGA 21MER (SEQ ID NO:81) R-GCTGCCTGAAGCACAAAAGG 20MER (SEQ ID NO: 82)P-CGCAGATCCTGCCAACCGAAGAGA 24MER (SEQ ID NO: 83) PARVALB.F-GACACCACTCTTCTGGAAAATGC 23MER (SEQ ID NO: 84) R-TTGCCAAACCAACACCTACCA21MER (SEQ ID NO: 85) P-ATCGGACACCACCTGTAGGGAGGACC 26MER (SEQ ID NO: 86)GAPDH F-CAGTGGCAAAGTGGAGATTGT 21MER (SEQ ID NO: 87)R-AATTTGCCGTGAGTGGAGTC 20MER (SEQ ID NO: 88)P-CCATCAACGACCCCTTCATTGACCTC 26MER (SEQ ID NO: 89)

Probes and primers were designed using Primer Express,(ABI-Perkin-Elmer). Guidelines for primer probe selection are includedin Williams and Tucker (1999) PCR applications, pp. 365-75 (AcademicPress).

B. Total RNA Preparation and Quantification

L4 and L5 were dissected from phosphate buffered saline perfused rats.Left and right sides were isolated in separate tubes. For total RNA usedin standard curves, all DRG were dissected from control rats. Total RNAwas isolated using the Qiagen Rneasy mini columns. Tissue washomogenized as per manufacturers instructions. Total RNA was quantifiedutilizing the Ribogreen Quantitaion Kit (Molecular Probes) and followingthe manufacturers instructions.

C. RT-PCR

Twenty five nanograms of total RNA was used per 50 ul reaction, exceptin standard curve reactions where 500, 250, 25 or 2.5 nanograms perreaction was used. Each reaction contained 25 pmol of eacholigonucleotide primer, 0.2 mM of each dNTP, 100 nM flourescentlylabelled oligonucleotide probe, 1×RT-PCR buffer (PE biosystems), 2.0 mMMgCl2, 20 U RNAse inhibitor, 12.5 MuLV reverse transcriptase (RT, PEbiosystems) and 2.5 U Amplitaq Gold polymerase (PE biosystems). Reversetranscription was performed for 30 min at 48 degrees C. followed by 95degrees C. for 10 min for Amplitaq Gold activation and RT inactivation,then PCR; 40 cycles of 95 degrees C. for 15 sec and 60 degrees C. forone and a half minutes.

D. Gene Expression Quantitation

Control RNA was used to generate standard curves for a housekeeping geneand the genes of interest with each taqman run. A standard curve wasobtained by plotting the threshold cycle (Ct) value obtained from theTaqman run versus the log of the quantity of control total RNA added.The resultant linear equation was solved for the log RNA value. Pluggingin the experimental Ct value produced the log of the experimental geneexpression value. Ten raised to the power of this value gives theexperimental gene expression in nanograms.

As can be seen from FIG. 14, pyridoxine treatment for two weeks resultedin a dose dependent decrease in neurofilament light chain (NFL), neuronspecific enolase (NSE), trkC, and calretinin expression. Both the dosedependency and magnitude of these decreases varies from marker tomarker, indicating a differential sensitivity of these proteins asmarkers of the neuronal damage.

In FIG. 15 the results of treating animals with two doses of Mab 2256along with the low dose (400 mg/kg daily) of pyridoxine are shown. NFLand NSE show a significant decrease in expression at this level ofpyridoxine treatment. Cotreatment of animals with 5 mg/kg of Mab 2256(subQ weekly) completely blocked this decrease in expression. A Mab 2256dose of 1 mg/kg had no appreciable effect on the expression of theseproteins. Neither trkC nor calretinin expression is significantlyaffected by this low dose pyridoxine treatment, but treatemtn with 5mg/kg Mab 2256 actually increases trkC expression over control level.

When animals are treated with the higher pyridoxine dose of 600 mg/kgdaily, the expression of NFL, NSE and calretinin falls to very lowlevels, while trkC expression falls to about 50% of control values (FIG.16). Cotreatment with Mab 2256 at either 1 mg/kg or 5 mg/kgsignificantly but not completely blocks the decrease in expression seenin trkC and calretinin. There is a slight trend towards protection seenwith NFL and NSE expression in animals treated with Mab 2256, but it didnot attain statistical significance. Thus, using multiple biochemicalmarkers of damage to large sensory neurons, Mab 2256 is seen to becapable of ameliorating the toxicity of pyridoxine treatment.

In order to examine the electrophysiolgical and behavioral effects ofpyridoxine neuropathy, rats were treated with twice daily injections of400 mg/kg pyridoxine for 8 days. The function of their large diametersensory afferents were tested electrophysiologically by recording theM-wave (direct motor) and H-wave (reflex sensory) response in themuscles of the foot after stimulation of the sciatic nerve at the thighand the calf (Gao et al. Ann. Neurol. 38(1):30-7 [1995]). Treatment withpyridoxine for 8 days resulted in a large decrease in the amplitude ofthe sensory response compared to the motor response as seen in FIG. 18.Cotreatment with Mab 2256 significantly blocked the pyridoxine-induceddecrease in the sensory amplitude. This is similar to effects publishedusing very high doses (20 mg/kg daily) of NT3 (Helgren et al., supra).

Animals treated with this regime of pyridoxine were also behaviorallytested for their proprioceptive function. They were trained to walkacross a horizontal ladder in order to escape a bright light and whitenoise stimulus into a dark box. The animals were videotaped from below,and the quality of the placement of their hindpaws on the rungs of theladder was read by an observer blind to their treatment. Each pawplacement was scored as a good placement (paw lands on forward part ofmetatarsals, immediately behind toes, with toes wrapping the rungimmediately), solid landing (paw hits other than immediately behindtoes, but solidly on rung, toes often not wrapping), near footfault (pawbarely hits rung, either on the extreme forward part of toes or rearwardaspect of heel, but does support weight) or footfault (paw either missesrung entirely or poor enough placement that foot does not support weightand falls through ladder upon weight bearing). Normal rats very quicklylearn to place their hindpaws correctly, which requires an excellentproprioceptive sense of where the hindpaw is in space. After treatmentwith pyridoxine (400 mg/kg twice daily for 8 days), the performance onthis task had declined, with an almost thirty percent decline in goodplacements and an increase in both footfaults and near footfaults (FIG.18). Cotreatment of the animals with Mab 2256 during this time, allowedthe animals to maintain a much higher degree of performance, with asmaller decline in good placements and smaller increases in footfaultsand near footfaults.

In summary, cotreatment with Mab 2256 ameliorates the toxic effects ofpyridoxine as measured biochemically, electrophysiologically, and byperformance on a behavioral task.

After establishing that the trkC Mabs were therapeutically at least aseffective as NT-3, the observed adverse event of hyperalgesia wasexamined. This side effect of NT-3 administration has been seen inrodents (see FIG. 19) and in humans (Chaudhary et al., Muscle and Nerve23:189-192 [2000]). Rats were trained and tested for thermal sensitivityof the hind paws using a Hargreaves device and then administered 1 mg/kgof Mab 2256 IV, or 1 mg/kg NT-3 subcutaneously in the scruff. At two,four, and six hours after administration, the rats were again tested fortheir thermal withdrawal times. As can be seen from FIG. 19, NT-3administration caused a significant heat hyperalgesia at four and sixhours post dosing, while the trkC Mab 2256 was without any effect onthermal pain sensation. So, at doses known to be effective in reversingor preventing neuropathyy, NT-3 does cause an increase in sensitivity topain, while the Mab 2256 does not.

Cisplatin, a widely used chemotherapeutic agent, induces a sensoryneuropathy with selective loss of vibration sense and proprioception.Here we demonstrate that neurotrophin-3 (NT-3), a member of the nervegrowth factor family of neurotrophic factors, restored to normal levelsthe reduced H-reflex-related sensory nerve conduction velocity inducedby cisplatin in rats. NT-3 treatment corrected an abnormal cytoplasmicdistribution of neurofilament protein in large sensory neurons in dorsalroot ganglia and the reduction in the numbers of myelinated fibers insural nerves caused by cisplatin. The NT-3-dependent reversal ofcisplatin neurotoxicity thus suggests the possible use of NT-3 in thetreatment of peripheral sensory neuropathy.

Chronic treatment of adult rats for 2-3 weeks with high doses ofpyridoxine (Vitamin B6) produced a profound proprioceptive loss, similarto that found in humans overdosed with this vitamin or treated with thechemotherapeutic agent cisplatin. Pyridoxine toxicity was manifest asdeficits in simple and precise locomotion and sensory nerve function andas degeneration of large-diameter/large-fiber spinal sensory neurons. Asassessed quantitatively in a beam-walking task and by EMG recording of Hwaves evoked by peripheral nerve stimulation, coadministration of theneurotrophic factor neurotrophin-3 (NT-3; 5-20 mg/kg/day, s.c.) duringchronic pyridoxine treatment largely attenuated the behavioral andelectrophysiological sequelae associated with pyridoxine toxicity.Furthermore, NT-3 administration prevented degeneration of sensoryfibers in the dorsal column of the spinal cord. These data areconsistent with the evidence that NT-3 is a target-derived neurotrophicfactor for muscle sensory afferents and suggest that pharmacologicaldoses of NT-3 may be beneficial in the treatment of large-fiber sensoryneuropathies.

Deposit of Biological Material

The following hybridoma cell lines and plasmids have been deposited withthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, USA (ATCC) on Jun. 21, 2000:

Hybridoma/Plasmid Designation ATCC No. 2.5.1 PTA-2151 6.1.2 PTA-21486.4.1 PTA-2150 2344 PTA-2144 2345 PTA-2146 2349 PTA-2153 2248 PTA-21472250 PTA-2149 2253 PTA-2145 2256 PTA-2152 DNA pXCA-2250HL PTA-2136 DNApXCA-2253HL PTA-2137 DNA pXCA-2256HL PTA-2138 DNA pXCA-6.1.2H PTA-2141DNA pXCA-6.4.1H PTA-2143 DNA pXCA-2345H PTA-2142 DNA pXCA-2349H PTA-2133DNA vegf4chim-6.1.2L PTA-2134 DNA vegf4chim-6.4.1L PTA-2135 DNAvegf4chim-2345L PTA-2139 DNA vegf4chim-2349L PTA-2140

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of the deposit. The organisms will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the cultures to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.12 with particular reference to 886 OG 638).

In respect of those designations in which a European patent is sought, asample of the deposited microorganism will be made available until thepublication of the mention of the grant of the European patent or untilthe date on which the application has been refused or withdrawn or isdeemed to be withdrawn, only by the issue of such a sample to an expertnominated by the person requesting the sample. (Rule 28(4) EPC)

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiments are intended to illustrate only certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that they represent. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and representative processes for their isolation, use, andmanufacture appear below, but should not be construed to limit theinvention.

All references cited throughout the specification and the referencescited therein are hereby expressly incorporated by reference.

1. A human anti-TrkC antibody selected from the group consisting ofantibodies 6.1.2 (ATCC No. PTA-2148), 6.4.1 (ATCC No. PTA-2150), 2345(ATCC No. PTA-2146), 2349 (ATCC No. PTA-2153), 2.5.1 (ATCC No.PTA-2151), and 2344 (ATCC No. PTA-2144).
 2. The antibody of claim 1selected from the group consisting of antibodies 6.1.2 (ATCC No.PTA-2148), 6.4.1 (ATCC No. PTA-2150), 2345 (ATCC No. PTA-2146), and 2349(ATCC No. PTA-2153).